HUPO 2005 Comparing Chromatography Columns for the Separation and ESI LC-MS/MS Analysis of Peptides and Protein Mixtures Hansjörg Toll1, Ulrike Schweiger-Hufnagel2, Dirk Wunderlich2, Catherine Stacey2, Christian G. Huber1; 1Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany; 2Bruker Daltonik GmbH, Bremen, Germany Introduction Results for Peptides Results for Proteins 10 tryptically digested proteins were separated on two For analyzing the detection sensitivity due to different tuning For ESI MS used for proteomic research, reversed-phase monolithic and a microparticular column, resp., and the MS parameter sets (Cyt C and Lys tuning), the signal-to-noise high-performance liquid chromatography (RP HPLC) is of data were compared in respect of: ratios for eight proteins were determined from outstanding relevance. This combination has made an chromatographic peaks, revealing significant differences in the enormous progress in the last decades: RP HPLC has Chromatogram Quality detectability (Figure 4). Moreover, spectrum quality also become more and more sensitive due to separation As shown in Figure 1, the 100 μm monolithic column showed depended significantly on the tuning parameters. With Lys performance and miniaturization while ESI MS has become the best separation, with slightly better patterning than the 200 tuning, which was optimized for highest sensitivity in the range faster and more sensitive. μm monolithic column and significantly better separation than of 1500-3000 m/z, all eight proteins were detected. With the This poster shows an enhancement of separation technique. the microparticular column, where individual peaks were Cyt C tuning, on the other hand, myoglobin, carbonic Monolithic columns based on poly-(styrene-divinylbenzene) sitting on top of a broad hump of unresolved background. anhydrase and cytochrome C were detected with significantly (PSDB) with a new very narrow inner diameter are compared higher sensitivity, while other proteins such as lysozyme or to standard microparticular columns. The progress in column Cumulative Ion Score transferrin remained undetected. As a consequence, it is condition has a positive effect on the complete instrumental Figure 2 summarizes the average cumulative ion scores strongly advisable to tune the mass spectrometer with a series setup. extracted from triplicate analyses. For all four different of proteins with different physico-chemical properties and to concentrations the highest score was achieved using the 100 run analyses with these different tunings in order to achieve Method μm monolithic column due to the highest number of identified maximum detection sensitivity for RP-HPLC-MS analysis of Peptide analysis peptides and the highest individual ion scores. complex protein mixtures. A mixture of 10 commercially available proteins was digested Peak Volume with trypsin according to standard protocols. Four different The peak volume which depends on column diameter and 1.5 ] amounts (Table 2) were injected onto three different columns: Cyt C Tuning .10-7 [counts peak dispersion was determined for 15 peptide peaks and Cah •PSDB based monolithic column 60 x 0.2 mm i.d. averaged. For monolithic columns (19.1 s and 19.4 s) it was •PSDB based monolithic column 60 x 0.1 mm i.d. 17 % smaller than for the microparticular column (23.1 s) •RP column with C18 silica particles 75 x 0.075 mm i.d. indicating a better separation performance of the monolithic Lac B LC and MS conditions are summarized in Table 1. columns. Myo Rib Cyt C 0 Parameter / 75 µm i.d. 100 µm i.d. 200 µm i.d. Reproducibility of Peptide Identification 0 10 20 signal intensity column Pepmap Monolith Monolith To investigate the reproducibility of peptide identifications for time [min] Instrument HCT (Bruker) combined to Ultimate (Dionex) all three separation columns, the number of peptides found 4.0 ] Lys Tuning .10-6 [counts Trf Flow rate A 200 nL/min 500 nL/min 2000 nL/min once, twice or three times in three consecutive runs was Cah Injection volume 100 nL 250 nL 1000 nL determined. As shown in Figure 3 the highest number of Lac A Lac B Linear gradient B 5-60% 0-40% 0-40% peptides that were identified in triplicate was achieved with the Gradient ACN in 0.05 % TFA 100 µm i.d. monolithic column, 24 % more than with the 75 µm Rib Column i.d. microparticulate column, and 53 % more than with the 200 Cyt C Lys Myo 55 °C temperature µm i.d. monolith. 0 Spray gas 15 psi 15 psi 20 psi 0 10 20 Dry gas 4 L/min Summary/ Conclusion for Peptides time [min] Spray voltage - 3500 V For peptides the 100 μm monolithic column Fig. 4. TIC MS of 8 proteins for Cyt C and Lys tuning. Ion charge control MS, MS/MS: 70.000 turned out to be most suitable. This is explained Full scan range 500-1500 m/z by the monolithic phase combined to a Summary/ Conclusion for Proteins MS/MS scan 200 - 2000 m/z remarkably small inner column diameter, leading For proteins monolithic columns are suited very Active exclusion 30 s, exclude after 1 spectrum Frag. Energy 1.5 V to sharp chromatographic peaks and high relative well, in particular when using multiple tuning No. Precursors 3 concentration of the eluting analytes. parameter sets. Smart frag. ramp 30 - 200 % 1.2 100 µm i.d. Table 1: Experimental LC and MS conditions A for constant linear flow velocity; B for comparable separation window Monolith 7000 signal intensity 10 -6 [counts] 100 μ m monolith amount amount 1 Protei amount 1/4 amount 1/2 6000 200 μ m monolith Organism 1/8 (fmol) n (fmol) (fmol) (fmol) 75 μ m Pepmap Cumulative Mascot Score CYT horse 80 160 320 640 5000 BSA bovine 37.5 75 150 300 . ß LAC bovine 55 110 220 440 4000 CAH bovine 34.5 69 138 276 CAT bovine 17.5 35 70 140 0 3000 LYS chicken 70 140 280 560 0 10 20 30 40 MYO horse 55 110 220 440 time [min] RIB A bovine 75 150 300 600 1.2 2000 TRF human 12.5 25 50 100 200 µm i.d. α LAC bovine 70 140 280 560 1000 Monolith signal intensity 10 -6 [counts] Table 2: Amount of the analyzed protein digests. For loading the respective sample amounts on three different 0 columns with different injection volumes, the sample amount 1 amount 1/2 amount 1/4 amount 1/8 concentrations were adjusted accordingly. Fig. 2. Average cumulative ion scores of peptides identified Protein analysis upon analysis of amounts 1, 1/2, 1/4, 1/8. . triplicate duplicate single Parameter / tuning Lys tuning Cyt C tuning 0 identification 140 mixture of 8 proteins (same as in Table 2 0 10 20 30 40 Sample except for BSA and catalase) time [min] 120 21 35 Flow rate 2 ul/ min 1.0 27 Injection amount 62.5 fmol per protein 75 µm i.d. Number of Peptides 100 21 Linear gradient 15-50% ACN in 0.05 % TFA Pepmap signal intensity Temperature 55 °C 25 10 -6 [counts] 80 43 Trap drive 179.1 93.2 Skimmer 150.0 V 34.4 V 60 Oct 1 12 V 12 V Oct 2 3.93 V 2.46 V 92 40 Octopole RF Amplitude 167.2 Vpp 88.5 Vpp 74 60 . Lens 1 - 1.7 V - 1.9 V 20 Lens 2 - 31.1 V - 36.1 V Cap exit 340 V 253.8 V 0 0 HV Capillary 3500 V 0 10 20 30 40 100 μ m i.d. 75 μm i.d. 200 μm i.d. Dry gas 4 L/min time [min] monolith Pepmap monolith Table 3: Sample, Experimental LC and MS conditions Fig. 1. TIC MS trace of the LC-MS/MS analysis of a peptide mixture using monolithic and microparticular columns. Fig. 3. Reproducibility of peptide identifications for three Sample amount 1/8. Experimental conditions are given in consecutive analyses with amount 1/4. Table 1.
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