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									             Nanoliter Solid Phase Extraction (SPE) Using Chromatographic Hollow Fibers for Sample
                                        Preparation for Mass Spectrometry
                                                              Mukta M.               Shukla
                                                                              Ashok K.     1,    Vladimir M.               Nelli I.                       Shukla1,                                                                                                                                                   Doroshenko 2,                                                                                                                                                                                                                                                                                                                        Taranenko2
                                                                    1Glygen Corp., 8990 Route 108, Suite C-1, Columbia, MD 21045, USA
                                                                 2MassTech, Inc., 6992 Columbia Gateway Drive, Columbia, MD 21046, USA


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Figure 2 we demonstrate the purification and partial separation of peptides from ovalbumin. The
                    INTRODUCTION                                                                                                                                     RESULTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         samples were eluted linear with 10ml of matrix solution and spotted 100nl per spot ( spot size was
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     less than a mm) at target plate and analyzed by AP-MALDI.
                                                                   In this poster, we have studied the purification and partial separation of peptides mixture after tryptic digest in chromatographic hollow fibers. The chromatographic material is only on the inner wall of the
The sample preparation techniques may be modified to
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       May18_003 #1 RT: 0.04 AV: 1 SM: 15B NL: 2.27E4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       T: + p ms [ 600.00-4000.00]

                                                                   capillary, therefore, there is hardly any or very little resistance or back pressure as compared to filled capillary HPLC columns or micro SPE cartridges. The fibers are made of inert materials such as Teflon                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      100

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improve the signal sensitivity and selectivity for mass            or polypropylene. Furthermore, fibers of any length can be used according to the specific application. These fibers are very economical as compared to capillary columns and can be used as disposable
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Fraction 5
spectrometric analysis. The sample preparation in micro            SPE and thus avoid cross contamination.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                75

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and nano-liters range without a loss of the sample is still a




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great challenge. Different methods for the sample                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         45

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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2285.0            2578.0




preparation in Nano-HPLC or Nano-LC-MS are known.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         30

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Here we describe the applications of the chromatographic           In Figure 1, we demonstrate the sample preparation (1ml) after tryptic digestion of fetuin and ovalbumin. The samples were eluted with step gradient and directly spotted on MALDI target plate.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       15

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          10                                            1403.5
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   2188.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2414.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2757.1
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         3947.6



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      3018.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           5              882.1 1037.3           1305.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               671.9


hollow fibers, where the chromatographic material is                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        800       1000      1200        1400       1600       1800         2000       2200
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                                                                   In Figure 2 we demonstrate the purification and partial separation of peptides from ovalbumin. The samples were eluted linear with 10ml of matrix solution and spotted 100nl per spot ( spot size was less                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          May18_011 #1 RT: 0.01 AV: 1 SM: 15B NL: 1.30E5


embedded on the inner walls of the capillary. These                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    T: + p ms [ 600.00-4000.00]
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                                                                   than a mm) at target plate and analyzed by AP-MALDI.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   95



chromatographic fibers are well suited for sample                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         90

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          85

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preparation for mass spectrometry in nano-liter range. The                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                75

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sample can be directly spotted at the atmospheric pressure




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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          50                                                                                            2282.0
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matrix-assisted laser desorption/ionization (AP-MALDI)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    45

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or MALDI- target plate or connected to mass spectrometer
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1917.1                         2413.4
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for electro spray ionization (ESI)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   1689.0                                                                                            3384.6              3711.6



                                                                                                                                                                                              Figure 1. AP-MALDI SPECTRA OF FETUIN AND OVALBUMIN
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1580.3                        2009.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   1403.9                                          2131.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           5                      1017.3                                                                                                2698.0 2902.1         3199.1            3562.5              3827.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               1304.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   712.5 882.4
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       May18_024 #1 RT: 0.02 1000 SM:1200 NL:1400
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        800      AV: 1 15B    3.28E5               1600       1800        2000       2200           2400       2600        2800       3000     3200     3400        3600       3800          4000



                                                                           Chromatographic Hollow Fiber ( LC-Fiber)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       T: + p ms [ 600.00-4000.00]
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                                                                                                                                                                                              (tryptic digest without purification and concentration)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2282.6
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          95

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          90



                    EXPERIMENTAL                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          85

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          80

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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   2413.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   Fraction 26
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Relative Abundance
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Materials                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 55

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          50

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          45

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          40

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Molecular Biology Grade Water from Biowittaker
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2578.0
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1917.1
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          20                                                               1760.5



(Walkersville, MD, USA) was used for the matrix and
                                                                                                                                                                                                                               May19_002 #1 RT: 0.04 AV: 1 SM: 15B NL: 3.08E2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             15                                                                                   2131.3                            2635.0
                                                                                                                                                                                                                               T: + p ms [ 600.00-4000.00]                                                                                                                                                                                                                                                                  May19_012 #1 RT: 0.02 AV: 1 SM: 15B NL: 1.38E3
                                                                                                                                                                                                                                                                                                1618.3                                                                                                                                                                                                                      T: + p ms [ 600.00-4000.00]                                                                                                                                                                                                                                                   10                                                                              2010.0
                                                                                                                                                                                                                                 100                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2902.1                3385.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             3384.5                                                                                                                                              1669.8
                                                                                                                                                                                                                                                                                                                                 2200.4                                                                                                                                                                                       100                                                                                                                                                                                                                                                                          5              883.1                    1403.8 1534.8                                                                        2822.6               3199.3             3565.8 3712.3          3947.9
                                                                                                                                                                                                                                  95                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             768.6             1052.9
                                                                                                                                                                                                                                                                                      1448.9                      1937.3                                                                                                                                                                                                                                                                                                                                                          2958.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           0


sample preparation solutions. Ovalbumin and fetuin
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               95
                                                                                                                                                                                                                                  90                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   800      1000 1200    1400     1600                    1800        2000       2200           2400       2600        2800       3000    3200      3400        3600       3800          4000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           1859.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               90                                                                                                                      2520.8                                                                                                                                          May18_030 #1 RT: 0.00 AV: 1 SM: 15B NL: 1.56E5
                                                                                                                                                                                                                                  85                                                                   1750.5                                                                                                                                                                                                                                                                                                                                                         2751.9                                                                                                                                                                                                                                 m/z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   3767.5                                                              T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                                                                                                                   2464.1                                                                                                                                                                      85                                                                                                                            2598.5
                                                                                                                                                                                                                                  80                                                                                       2081.6      2247.8                                                                        3562.9                                                                                                                                                                                                                                               2807.1                                                                                                                                                                                                 1860.3
                                                                                                                                                                                                                                                                                                                                                                        2807.9                                                                                                                                                                                                                                                                                                                                                                                                                           100



were obtained from Sigma. The matrix material, α-
                                                                                                                                                                                                                                                                                                            1756.4                                                                                                                                                                                                                                                                                                                                                                                                                            3944.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               80                                                                                                                                                                 3212.5
                                                                                                                                                                                                                                  75                                                                                                                                              2979.3                                                                                                                                                                                                                                               2282.0
                                                                                                                                                                                                                                                                               1434.5                                                                                                                                                                                                                                                                                                                                                                                                 3025.9                                 3695.3                                                                       95
                                                                                                                                                                                                                                                                                                                                             2374.8               2788.0                                                                                                                                                       75                                                                                                                                                                                                         3910.8
                                                                                                                                                                                                                                  70                          1072.0                                                                                                                                                                                                                                                                                                                                                                           2411.8
                                                                                                                                                                                                                                                                                                                                                                                                      3325.1                                                                                                                                                                                                                                                                                3129.8                        3624.4                                                                          90
                                                                                                                                                                                                                                                                  1169.5                                                                                                                                                                                                                                                       70
                                                                                                                                                                                                                                  65                                                                                                                                                      3087.0                                                                                                                                                                                                                         2087.3
                                                                                                                                                                                                                                                                              1330.6                                                                                                                                           3737.1                                                                                                                                                                                                                                                                                                                                                                     85
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               65     718.9

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                                                                                                                                                                         Relative Abundance
                                                                                                                                                                                                                                  60               903.5                                                                                                                                                                                                                                                                                                                                                        1882.8
                                                                                                                                                                                                                                                                                                                                                                                                            3442.0                          3956.1


cyano-4-hydroxycinnamic acid (4-HCCA) was
                                                                                                                                                                                                                                                                                                                                                               2759.4                                                                                                                                                                                                           1402.4                                                                                                                                                                                                                    80




                                                                                                                                                                                                                                                                                                                                                                                                                                                        Relative Abundance
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               60                                                                     1785.9        2066.4
                                                                                                                                                                                                                                  55                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2282.9
                                                                                                                                                                                                                                                                                                                                                            2619.6                                                                      3924.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     947.8                                                                                                                                                                                                                                                75
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               55                                                                 1703.4
                                                                                                                                                                                                                                  50    717.3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               50                                                         1564.8
                                                                                                               Chromatographic media is embedded in the                                                                           45
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1363.0                                                                                                                                                                                                                        65


obtained from Fluka (Buch, Switzerland). Trypsin
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               45                               1159.6
                                                                                                                                                                                                                                  40                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2413.9
                                                                                                               inner surface of the capillary.




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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          60
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               40                                                                                                                                                                                                                                                                                                                                                             2089.9
                                                                                                                                                                                                                                  35                                                                                                                                                                                                                                                                                                           931.6                   1327.3                                                                                                                                                                                                                             55
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               35                          1033.8
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            838.3                                                                                                                                                                                                                                                         50
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beads (Poroszyme Bulk Immobilized Trypsin,                                                                                                                                                                                        25

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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2009.9


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2578.4
                                                                                                                                                                                                                                  10


PerSeptive Biosystems) was obtained from Applied                                                                                                                                                                                   5

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                                                                                                                                                                                                                                             800          1000         1200          1400       1600       1800        2000         2200         2400       2600         2800        3000      3200         3400        3600        3800         4000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               10

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                5

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          30

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          25

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               1534.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           1760.5

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2635.4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       3384.8

                                                                                                                                                                                                                                                                                                                                           m/z                                                                                                                                                                                               800        1000          1200       1400          1600      1800        2000        2200           2400         2600        2800       3000          3200         3400       3600        3800          4000                                                  15


Biosystems (Foster City, CA, USA). Different affinity                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    m/z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          10    713.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          882.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1403.8                                         2129.4                              2642.8 2903.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   2960.3




                                                                                                  LC-Fiber
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1017.5         1346.6                                                                                                                    3441.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2756.6              3199.1                          3769.1    3947.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           5

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           0


materials mini columns (TopTip) were obtained and                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       800       1000      1200        1400       1600       1800         2000       2200
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              m/z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     2400       2600        2800      3000     3200      3400       3600        3800         4000




embedded (NuTip) in the micropipette tips by using
Glygen (Columbia, MD, USA) Technology.



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       May18_031 #1 RT: 0.01 AV: 1 SM: 15B NL: 2.52E4

Mass Spectrometry                                                    LC -Fiber
                                                                                                                                                                                                                                                May19_005 #1 RT: 0.01 AV: 1 SM: 15B NL: 2.13E5
                                                                                                                                                                                                                                                T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                   100
                                                                                                                                                                                                                                                                                          1475.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    May19_015 #1 RT: 0.02 AV: 1 SM: 15B NL: 5.78E4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 2282.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 1860.1


                                                                                                                                                                                                                                                     95

                                                                                                                                                                                                                                                     90
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        95
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          95

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          90                                                                                                                           Fraction 36
                                                                     Capillaries of different i.d. and length are made with the chromatographic particles directly
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        90


Experiments were carried out on a Thermo Finnigan (San Jose,                                                                                                                                                                                         85

                                                                                                                                                                                                                                                     80
                                                                                                                                                                                                                                                                                                                        0.1%TFA in 2% ACN
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        85

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      0.1%TFA in 2% ACN                                                                                                                                                                                   85

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          80

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          75


CA, USA) LCQ Deca XP ion trap mass spectrometer integrated           attached to the inner surface of the wall of the capillary. Chromatographic particles of five or                                                                                75

                                                                                                                                                                                                                                                     70

                                                                                                                                                                                                                                                     65                                                                                                                                            2892.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        75

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        70

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        65
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2413.8                                                                                                                                          70

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          65                                                   1534.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2089.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2009.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           1774.8




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Relative Abundance
                                                                     ten micron diameter are used. The chromatographic materials such as C-18, polymer-based                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              60
                                                                                                                                                                                              Relative Abundance




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Relative Abundance
                                                                                                                                                                                                                                                     60                                                                                                                                                                                                                                                                                 60                                                                                                                                                                                                                                                                                                                                                                                    2578.4

with an AP/MALDI ion source (MassTech Inc., Columbia, MD,                                                                                                                                                                                            55

                                                                                                                                                                                                                                                     50
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        55

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        50                                                                            1860.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          55

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          50
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2282.1                                                         3384.6




USA). The AP/MALDI source is described in detail in ref. [8-         (reverse phase) and carbon particles are attached to the inner wall of the capillary. The                                                                                       45

                                                                                                                                                                                                                                                     40
                                                                                                                                                                                                                                                                                                                                                    2121.3

                                                                                                                                                                                                                                                                                                                                                                                 2577.9
                                                                                                                                                                                                                                                                                                                                                                                                                         3250.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        45

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           1688.8


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           2237.5                                         2902.9                       3384.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            3712.9

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               3769.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          45

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          40

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          35
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                713.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          882.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               2339.3
                                                                                                                                                                                                                                                     35                                                                                                                                                                                                                                                                                 35                                                                                                                       2471.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     2413.8


10], but our specific arrangement is outlined here. A Thermo         capillaries are made of polypropylene or Teflon. The peptide mixture from tryptic digest such                                                                                   30

                                                                                                                                                                                                                                                     25
                                                                                                                                                                                                                                                                                                                  1519.1                                                                                3006.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        30

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        25
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1403.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2578.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2960.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          30

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          25
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   768.3          1017.3


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         1508.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2635.1
                                                                                                                                                                                                                                                     20                                                                                                                                                                                                                                                                                                                                                                                 2189.5                                                                                                       3827.0                                               20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        20                                             1346.6         1580.4                                                              2642.9                                                                                                                                                                                                                    2146.4

                                                                     as ovalbumin and fetuin were purified by chromatographic hollow fibers and were analyzed
                                                                                                                                                                                                                                                                                                                                             2018.0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           3199.9
                                                                                                                                                                                                                                                                                                                                                                         2463.4

Laser Science Inc. (Franklin, MA, USA) Model 337 Si nitrogen                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        3017.8                                                                                                15                                            1475.8                                                                            2756.1          3104.5           3441.6
                                                                                                                                                                                                                                                     15                                                              1621.9                                                                                    3018.5                                                                                                                   15                                                                                   1898.9                                                                                                                                                                                                                                                                                                                                                  3563.8
                                                                                                                                                                                                                                                                                                       1461.9                                                                                                                                                                                                                                                                                                                                                                     2756.8                                                                                                                                              1051.5                                                                                                                                                    3826.0
                                                                                                                                                                                                                                                                                             1270.4                                                                                                                                                                                                                                                                                                                                                                                                          3199.8                                                                                                                            1226.8                                                                                                             3223.1
                                                                                                                                                                                                                                                     10                                                                             1841.9                         2349.8                                      3182.5                                                                                                                                                            1248.4                                               2130.4                                                                                3442.1                                                                        10
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        10                                                                                                                                                                                                                                                                                                                                                                                                                                                              3912.1
                                                                                                                                                                                                                                                                  774.8 883.0               1253.1 1281.3                                                                             2683.8                                                     3627.1 3740.8                                                                                               882.8    1052.0
                                                                                                                                                                                                                                                      5                                                                                                                                                                        3273.4                                                          3947.1                                                                                                                                                                                                                                                                                                      5


                                                                     by HPLC and AP-MALDI. For the mass spectrometric analysis, samples are prepared on the                                                                                                   713.5                                                                                                                                                                                                                                                                      5            845.1


UV laser was used. Its wavelength was =337 nm, laser pulse                                                                                                                                                                                            0
                                                                                                                                                                                                                                                                       800       1000          1200       1400       1600        1800        2000          2200
                                                                                                                                                                                                                                                                                                                                                                  m/z
                                                                                                                                                                                                                                                                                                                                                                        2400      2600        2800       3000         3200         3400      3600                            3800                                  4000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        800          1000       1200       1400         1600        1800        2000        2200
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    m/z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2400       2600        2800       3000           3200        3400       3600          3800       4000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       800        1000      1200        1400       1600       1800        2000       2200
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              m/z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2400        2600        2800      3000     3200     3400        3600       3800          4000




duration was about 4 ns, and the laser beam was focused to           gold-plated target plate by mixing aliquots of 1.0 µl of the purified peptide mixture with 1.0 µl                                                                             May19_006 #1 RT: 0.02 AV: 1 SM: 15B NL: 2.26E5
                                                                                                                                                                                                                                                   T: + p ms [ 600.00-4000.00]                                                                                                                                                                                                                                                      May19_017 #1 RT: 0.03 AV: 1 SM: 15B NL: 3.03E4
                                                                                                                                                                                                                                                                                                                                                                                                                          3251.4                                                                                                    T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                     100


approximately 500 mm size spot. The maximum laser energy             of matrix and dried at room temperature. Matrix composition was 1.15 mg/ml of 4-HCCA                                                                                             95

                                                                                                                                                                                                                                                      90
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      100

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       95
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 2282.1

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2413.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       3384.9              3712.3



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       90


on the target was measured to be about 140 J/pulse, using a          dissolved into a solution of 70% ACN and 0.1% TFA. The instrumental setup consisted of the                                                                                       85

                                                                                                                                                                                                                                                      80

                                                                                                                                                                                                                                                      75
                                                                                                                                                                                                                                                                                            0.1%TFA in 5% ACN                                                                                                                                                                                                                          85

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    0.1%TFA in 5% ACN                                                                                 2578.1
                                                                                                                                                                                                                                                                                                              1476.0

                                                                     AP UV-MALDI ion source equipped with a 10 Hz nitrogen laser, and integrated with LCQ-
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       75
                                                                                                                                                                                                                                                      70

Molectron Detector (Portland, OR, USA) Model EM-400 laser                                                                                                                                                                                             65
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       70

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       65
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1860.1

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Discussion
                                                                                                                                                                                                         Relative Abundance




                                                                                                                                                                                                                                                      60


                                                                     Deca-XP ion trap mass spectrometer.




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Relative Abundance
                                                                                                                                                                                                                                                                                                                    1519.3                                                                                                                                                                                                             60


energy meter. During operation, the laser energy was attenuated
                                                                                                                                                                                                                                                      55
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         2903.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       55                                                                                                                                                                                                                 3947.9
                                                                                                                                                                                                                                                      50
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       50                                                                                                                                  2699.8
                                                                                                                                                                                                                                                      45                                                                                                                                                                                                                                                                                                                                                   1688.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       45

to the level of about 60 J/pulse. Sample and laser spots were                                                                                                                                                                                         40

                                                                                                                                                                                                                                                      35

                                                                                                                                                                                                                                                      30
                                                                                                                                                                                                                                                                                                                                                                            2463.5
                                                                                                                                                                                                                                                                                                                                                                                                            3006.5

                                                                                                                                                                                                                                                                                                                                                                                                                3018.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       40

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       35
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2237.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2959.8

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       The Chromatographic Hollow Fiber ( LC-Fiber) has following
                                                                                                                                                                                                                                                                                                                                                                                                     2892.9

observed on a TV monitor at a viewing angle of 450 with a total                                                                                                                                                                                                                                                                                         2121.1                                                                                                                                                                         30
                                                                                                                                                                                                                                                      25                                                                                                                                                                                                                                                                                                                                                                                                                          2757.0                                   3441.6
                                                                                                                                                                                                                                                                                                                                                                                                                     3206.0                                                                                                            25                                                                                               2189.1                                                               3199.9
                                                                                                                                                                                                                                                      20

                                                                                                                                                                                                                                                      15
                                                                                                                                                                                                                                                                                                                       1621.9
                                                                                                                                                                                                                                                                                                                                                                                  2577.6
                                                                                                                                                                                                                                                                                                                                                                                       2683.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1403.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1580.3                       2131.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1916.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  3018.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 3366.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       advantages:
magnification of about 100x using a CCD camera.                                                                                                                                                                                                       10

                                                                                                                                                                                                                                                          5       774.6
                                                                                                                                                                                                                                                                             882.9 996.9
                                                                                                                                                                                                                                                                                                  1270.4
                                                                                                                                                                                                                                                                                                         1281.1
                                                                                                                                                                                                                                                                                                                                1781.8
                                                                                                                                                                                                                                                                                                                                                 2018.0     2214.1
                                                                                                                                                                                                                                                                                                                                                                     2429.9
                                                                                                                                                                                                                                                                                                                                                                                             2710.6
                                                                                                                                                                                                                                                                                                                                                                                                                 3189.6
                                                                                                                                                                                                                                                                                                                                                                                                                                3274.1 3423.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                        3741.4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   3975.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       15

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       10

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        5      712.8 882.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1051.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             1165.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1346.4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               2023.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3654.4



                                                                                                                                                                                                                                                          0
                                                                                                                                                                                                                                                                        800          1000       1200       1400        1600         1800         2000       2200
                                                                                                                                                                                                                                                                                                                                                                   m/z
                                                                                                                                                                                                                                                                                                                                                                         2400        2600      2800         3000        3200        3400         3600                         3800                                  4000                0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       800          1000       1200       1400         1600      1800          2000        2200           2400         2600        2800        3000          3200       3400        3600       3800           4000                                    *No Contamination due to solid support
Operation                                                                                                                                                                                                                                          May19_008 #1 RT: 0.03 AV: 1 SM: 15B NL: 4.35E5
                                                                                                                                                                                                                                                   T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    May19_018 #1 RT: 0.00 AV: 1 SM: 15B NL: 9.22E4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   m/z




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      *Separation in few nanoliters
                                                                                                                                                                                                                                                                                                                                                        2121.5                                                                                                                                                                                                                                             1689.3
                                                                                                                                                                                                                                                     100                                                                                                                                                                                                                                                                              100
                                                                                                                                                                                                                                                      95                                                                                                                                                                                                                                                                               95

Experiments were carried out in a repetitive laser shot mode                                                                                                                                                                                          90

                                                                                                                                                                                                                                                      85
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       90

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       85



(frequency 10 Hz).       The laser trigger times were not
                                                                                                                                                                                                                                                      80

                                                                                                                                                                                                                                                      75

                                                                                                                                                                                                                                                      70
                                                                                                                                                                                                                                                                                                                                                             0.1%TFA in 25% ACN
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       80

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       75

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               0.1%TFA in 25% ACN                                                                      3385.4                                                                         *Fiber length can be between 1-50cm
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3770.1
                                                                                                                                                                                                                                                      65

synchronized with the LCQ operation. Operating conditions for                                                                                                                                                                                                                                                                                                                                                                                                                                                                          65
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1860.5                                                                                                               3713.0
                                                                                                                                                                                                         Relative Abundance




                                                                                                                                                                                                                                                      60
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Relative Abundance




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      *Internal diameter can be 50-500 microns
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       60                                                                                                        2284.1
                                                                                                                                                                                                                                                      55                                                                                                                                                                  3251.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       55
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2414.4

all full MS and MS/MS experiments were as follows: automatic                                                                                                                                                                                          50

                                                                                                                                                                                                                                                      45

                                                                                                                                                                                                                                                      40
                                                                                                                                                                                                                                                                                                              1476.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       50

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       45

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         2642.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     3827.4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           3948.1




gain control (AGC) was off, ion injection time was 220 ms, the                                                                                                                                                                                        35

                                                                                                                                                                                                                                                      30
                                                                                                                                                                                                                                                                                                                                                                                                            3006.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       35

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       30                                                 1404.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             1890.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2471.3                                                                                                                                *Minimal sample loss
                                                                                                                                                                                                                                                      25                                                                                                                                                                                                                                                                               25                                                                                                                                                                                   3417.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1580.8                  1899.0                                              2756.5

temperature of the LCQ input capillary was held at 280 C.                                                                                                                                                                                             20

                                                                                                                                                                                                                                                      15                                         1253.5
                                                                                                                                                                                                                                                                                                                    1519.3
                                                                                                                                                                                                                                                                                                                                                            2213.9
                                                                                                                                                                                                                                                                                                                                                                            2463.4

                                                                                                                                                                                                                                                                                                                                                                                                     2893.1
                                                                                                                                                                                                                                                                                                                                                                                                                     3206.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       20

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       15
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            883.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1347.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2023.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2238.3


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2189.4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         2903.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2960.6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             3199.5



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 3367.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                3442.1

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 3531.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Still the plumbing of these fibers is a challenge due to small
                                                                                                                                                                                                                                                      10                                                                                                                          2577.8                         3189.1         3262.8                                                                                                 10                                        1305.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1052.5                                                                                                                         3143.3

Typical high voltage applied to the target plate was 2000 V.                                                                                                                                                                                              5

                                                                                                                                                                                                                                                          0
                                                                                                                                                                                                                                                                 713.6

                                                                                                                                                                                                                                                                        800
                                                                                                                                                                                                                                                                             883.1    1052.4


                                                                                                                                                                                                                                                                                     1000       1200
                                                                                                                                                                                                                                                                                                         1462.1



                                                                                                                                                                                                                                                                                                           1400        1600
                                                                                                                                                                                                                                                                                                                             1699.9



                                                                                                                                                                                                                                                                                                                                    1800
                                                                                                                                                                                                                                                                                                                                              2001.4



                                                                                                                                                                                                                                                                                                                                                 2000       2200
                                                                                                                                                                                                                                                                                                                                                                     2398.9



                                                                                                                                                                                                                                                                                                                                                                         2400
                                                                                                                                                                                                                                                                                                                                                                                       2683.9


                                                                                                                                                                                                                                                                                                                                                                                     2600      2800         3000        3200
                                                                                                                                                                                                                                                                                                                                                                                                                                    3423.0


                                                                                                                                                                                                                                                                                                                                                                                                                                    3400         3600
                                                                                                                                                                                                                                                                                                                                                                                                                                                        3741.0



                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3800
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3975.3


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    4000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        5

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                713.6



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       diameters and pressure.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       800           1000       1200       1400         1600        1800       2000        2200           2400          2600       2800        3000           3200      3400         3600          3800       4000


Spectra were averaged over a 30 s to 3 min collection time. The                                                                                                                                                                                    May19_010 #1 RT: 0.04 AV: 1 SM: 15B NL: 3.55E5
                                                                                                                                                                                                                                                                                                                                                                   m/z



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     May19_019 #1 RT: 0.04 AV: 1 SM: 15B NL: 1.04E5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    m/z



                                                                                                                                                                                                                                                   T: + p ms [ 600.00-4000.00]

LCQ was operated in high mass range mode (up to 4000 Da),
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     T: + p ms [ 600.00-4000.00]
                                                                                                                                                                                                                                                                                               1519.4                                                                                                                                                                                                                                                                                                                                                                  2578.3                                                               3712.6
                                                                                                                                                                                                                                                      100                                                                                                                                                                                                                                                                              100
                                                                                                                                                                                                                                                      95                                                                                                                                                                                                                                                                                95



and was calibrated using the AP/MALDI source and a standard                                                                                                                                                                                           90

                                                                                                                                                                                                                                                      85

                                                                                                                                                                                                                                                      80                                                                      0.1%TFA in 50% ACN                                                                          3251.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        90

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        85

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             0.1%TFA in 50% ACN                       1860.5                      2283.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2414.3



protein. Mass spectra were obtained by averaging 50–200                                                                                                                                                                                               75

                                                                                                                                                                                                                                                      70

                                                                                                                                                                                                                                                      65
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        75

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        70

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        65


individual laser shots.
                                                                                                                                                                                                          Relative Abundance




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Relative Abundance




                                                                                                                                                                                                                                                      60                                                                                                                                                                                                                                                                                60
                                                                                                                                                                                                                                                      55                                                                                                                                                                                                                                                                                55
                                                                                                                                                                                                                                                      50                                                                                                                                                                                                                                                                                50
                                                                                                                                                                                                                                                                                                                                                        2121.3




Sample Preparation
                                                                                                                                                                                                                                                      45

                                                                                                                                                                                                                                                      40

                                                                                                                                                                                                                                                      35
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        45

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        40

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        35
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           2642.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2700.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           2903.6


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               2960.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        3385.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     3826.3

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Conclusion
                                                                                                                                                                                                                                                      30                                                   1476.0                                                                                                                                                                                                                       30
                                                                                                                                                                                                                                                                                                                                                                                                            3007.0
                                                                                                                                                                                                                                                                                                                                                                            2463.5
                                                                                                                                                                                                                                                      25                                                                                                                                                                                                                                                                                25                                                                                                                                         2756.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1760.5       1900.0                                                                                                                        3949.1
                                                                                                                                                                                                                                                      20                                                                                                                                                                                                                                                                                                                                   1403.9

Ovalbumin and fetuin were digested directly in a volatile buffer
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           1689.3              1917.5
                                                                                                                                                                                                                                                      15                                                                                                                                                        3018.4                                                                                                                  15                                                        1523.6                                   2237.5                                                   3017.6                  3442.3
                                                                                                                                                                                                                                                                                                                                                            2214.1                                                                                                                                                                                                                     1346.9                                   2022.8
                                                                                                                                                                                                                                                                                                 1253.4                1590.6                                                                        2893.0                                                                                                                                                                                                                                                                                                   3198.6
                                                                                                                                                                                                                                                      10                                                                                                                          2577.5                         3189.3         3268.4                                                                                                  10
                                                                                                                                                                                                                                                                             882.8                                               1789.1       1996.4                2399.0                  2683.9                                                    3741.5
                                                                                                                                                                                                                                                                                                         1320.1

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       The LC-Fiber can be used as:
                                                                                                                                                                                                                                                                                       1074.0                                                                                                                                      3423.1

containing 100 mM ammonium bicarbonate. 3.0 µl of 1 mM                                                                                                                                                                                                    5

                                                                                                                                                                                                                                                          0
                                                                                                                                                                                                                                                                   769.0


                                                                                                                                                                                                                                                                        800          1000       1200       1400        1600         1800         2000       2200
                                                                                                                                                                                                                                                                                                                                                                  2349.8


                                                                                                                                                                                                                                                                                                                                                                         2400        2600
                                                                                                                                                                                                                                                                                                                                                                                              2875.3


                                                                                                                                                                                                                                                                                                                                                                                               2800         3000        3200        3400
                                                                                                                                                                                                                                                                                                                                                                                                                                                  3646.0


                                                                                                                                                                                                                                                                                                                                                                                                                                                 3600
                                                                                                                                                                                                                                                                                                                                                                                                                                                             3976.1


                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3800                                  4000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         5

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   713.4

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        800
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             882.5    1051.6


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 1248.3


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1200        1400        1600        1800        2000        2200             2400       2600        2800          3000        3200        3400       3600          3800       4000
                                                                                                                                                                                                                                                                                                                                                                   m/z                                                                                                                                                                                                                                                                                 m/z


CaCl2 was added to adjust the pH to 8.5. To enhance the
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       *HPLC-loop for online sample prep.
recovery of hydrophobic peptides, 5 % acetonitrile was added.
The digestion was carried out by using 10 µl trypsin beads with                                                                                                                                                                                                                                                                                    A                                                                                                                                                                                                                                                                                                    B                                                                                                              *Nano and micro SPE for sample cleanup before injecting the
1:100 v/v enzyme to substrate ratio. Digestion was carried out                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         sample in micro or nano HPLC.
at +37oC, with overnight shaking. Then, the glycoprotein
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       *Capillary Electro-Chromatography.
samples were spun at 15,000 rpm for 3 min. and the supernatant
was removed. The peptides generated in the supernatant were                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            *Sample enrichment by using different chromatographic media
analyzed by AP-MALDI MS.                                                                                                                                                                                                        Figure 1, we demonstrate the sample preparation (1ml) after tryptic digestion of fetuin (A)                                                                                                                                                                                                                                                                                                                                                                                                                                            such as C-18, ion-exchange, polymer based, HILIC and
                                                                                                                                                                                                                                and ovalbumin (B). The samples were eluted with step gradient and directly spotted on                                                                                                                                                                                                                                                                                                                                                                                                                                                  immobilized enzyme, Protein A in LC-Fiber.
                                                                                                                                                                                                                                MALDI target plate.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    *Atmospheric pressure matrix- assisted laser desorption/ionization
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       (AP-MALDI) mass spectrometry in combination with LC-Fiber is
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       particularly beneficial for peptide analysis because of minimal
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       fragmentation of analyte ions, large tolerance to the laser energy and
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       the ability to produce primarily singly-charged ions.

								
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