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					Introduction to 2D LC-
         MS/MS
         (Yuanming Luo)
    Institute of Microbiology
   Chinese Academy of Sciences
Fully integrated 2D-LC/ion trap MS
              Hardware Improvement
          ---- New Orthogonal Ion Source



                                 New Endcap Electrodes


                                 Entrance Lens

                       Square Quadrupole




                                      New Inter-Octapole Lens
Attomole Sensitivity !!!
1D-strong cation exchange
  column (Biobasic SCX)
Pressure cell
Xcalibur-control the instrument
Bioworks 3.1-database search software
    package containing SEQUEST
   Application of 2D LC-MS/MS
 Molecular weight determination

 2D gel spots (especially the spots that can’t be
  identified by PMF analysis)

 Protein complex (after primary factionation)

 Proteome separation and identification

 Multi-dimensional liquid chromatography MS-based
  differential proteomics

 Quantitative proteomics (including ICAT or stable
  isotope labeling-based differential proteome
  analysis)
Molecular weight determination
   of myoglobin by BIOMASS
         Calculation
Mr:16951.38+
    /-0.33
High throughput
       gel spot
       analysis
Tandem RP Columns
Automated Protein Identification of 2-D gel
                   spots
       Sensitivity and Throughput !!!


                          Digest
                ?


          SEQUEST
      Cross-Correlation
         Comparison
                                    Protein identified
 High throughput gel spot
         analysis
1. Protein mixture is separated by 2D
   gel electrophoresis
2. Excise target gel spot
3. Perform in-gel digestion with
   trypsin.
4. Extract peptides from gel spot.
5. Run peptide mixture with ProteomeX
   in 1D High Throughput mode.
                      Analysis of 2D Gel Spots Using ProteomeX
                               High Throughput Method
RT: 0.00 - 102.10
                                                                                                                                69.67                                                NL: 2.39E7
        100                    Spot 1                                      Found t-PA                                                                                                Base Peak F: + c Full ms [
                                                                                                                                                                                     300.00-2000.00] MS
                     50                                                                           51.42                                 74.38                                        GelSpot_tPA1_C1
                                                         22.49                                                                                   75.40
                                 10.92        13.98               28.29             42.27                               66.11                                82.75        97.56
                      0
                                                                                                                  61.66                                                              NL: 9.02E6
        100

                     50
                                         2
                                     13.77    19.53
                                                      21.95
                                                                  28.64     41.22       45.20
                                                                                                  51.90                 65.73      70.49
                                                                                                                                                   82.59        83.90
                                                                                                                                                                                     Base Peak F: + c Full ms [
                                                                                                                                                                                     300.00-2000.00] MS
                                                                                                                                                                          100.31     gelspot_tpa2_c2

          0                                                                                                                                                                          NL: 1.16E7
                                                          22.68
        100
                                             3                             Found t-PA
                                                                                    51.85                                                       79.67
                                                                                                                                                                                     Base Peak F: + c Full ms [
                                                                                                                                                                                     300.00-2000.00] MS
                                                              23.16                                                     70.58     72.35
                     50                           22.05                   35.66        40.14                                                              82.29                      gelspot_tpa3_c1
                              3.68      17.20                                                                                                                           95.10
                      0
                                                                                                                  61.76                                                              NL: 1.41E7
Relative Abundance




        100                                                                                                                                                                          Base Peak F: + c Full ms [

                     50
                                             4        21.83
                                                                  34.16    36.77        43.07
                                                                                                  51.89
                                                                                                                         65.77     70.29         80.86
                                                                                                                                                                                     300.00-2000.00] MS
                                                                                                                                                                                     gelspot_tpa4_c2
                              0.48     8.82      20.55                                                                                                     82.44          100.61
          0                                                                                                                                                                          NL: 2.11E7
                                                                                                                       63.93
        100
                                             5
                                                          23.06
                                                                  29.58
                                                                                  Found t-PA
                                                                               39.32
                                                                                                                                                                                     Base Peak F: + c Full ms [
                                                                                                                                                                                     300.00-2000.00] MS
                     50                                                                           51.72                                                                              gelspot_tpa5_c1
                                                 17.44                    35.12     42.52                    59.20         65.88                79.87     82.27
                              4.34     15.18                                                                                                                              97.30
                      0
                                                                                                                  61.60                                                              NL: 1.15E7
        100

                     50
                                             6        21.90
                                                  19.62     27.77           37.34        48.81
                                                                                                  51.85                         70.37
                                                                                                                                           72.64        81.26
                                                                                                                                                                                     Base Peak F: + c Full ms [
                                                                                                                                                                                     300.00-2000.00] MS
                                                                                                                                                                                     gelspot_tpa6_c2
                              1.26      12.70                                                                                                                     85.70    98.74
          0                                                                                                                                                                          NL: 8.00E6
                                                          22.74                                   51.83
        100

                                        11.13
                                             7    21.28           29.56        44.47        48.00         54.56
                                                                                                                       63.79     70.37          79.86
                                                                                                                                                     81.19
                                                                                                                                                                 84.87
                                                                                                                                                                                     Base Peak F: + c Full ms [
                                                                                                                                                                                     300.00-2000.00] MS
                     50        8.54                                                                                                                                        92.89     gelspot_tpa7_c1

                      0
                          0              2            4               6            8              10              12              14              16              18            20
                                                                                               Time (min)
Global Protein Identification
             Global Protein
             Identification
                                                                    Reverse column
                                      SCX column fractionation        separation




Protein mixture   Protein digests




                                                                                     Auto MS/MS detection


   Results
                                                                 Tandem MS spectra
                       BioWorks data base search
Plumbing Diagrams for
    Proteome X.
                   2D-RP2 column
      1D-SCX
      column




   2D-RP1 column
        Global Protein
        Identification
1. Extract proteins from cell lysates

2. Reduce proteins to peptide fragments
  by tryptic digestion.

3. Analyze peptide mixture by 2D LC-
  MS/MS with ProteomeX.

4. Peptide and proteins identified by
  TurboSEQUEST software.
Protease Digestion of
       Proteins
1D LC-MS/MS of proteins from
      A431 cell lysates
RT: 0.00 - 600.00
            33.95                                                                                                                                     NL:
           431.84                                                                                                                                     2.28E9
 100                                                                                                                                                  Base Peak
                                                                                                                                                      MS
                                     35.12                                                                         30 min gradient                    A431_30min
                      50            1163.80                                                                                                           G_1029

                      0                                                                                                                               NL:
                                26.66
                               652.24                                                                                                                 2.65E9
          100                                                                                                                                         Base Peak
                                    42.92                                                                                                             MS
                                   1138.32
                      50                      76.43                                                                     60 min                        a431_60min
                                                                                                                                                      g_1029
                                              667.61
 Relative Abundance




                      0                                                                                                                               NL:
                                       68.71
                                50.66 1160.53                                                                                                         1.28E9
          100                   486.92             117.86                                                                                             Base Peak
                                                   563.10 138.63                                                                                      MS
                      50
                                                          703.32                                                       120 min                        a431_120mi
                                                                                                                                                      ng_1029
                                                              148.78
                                                              371.00
                      0                                                                                                                               NL:
                           14.27
                           344.05             84.72                200.37                                                                             1.49E9
          100                                1154.55               563.16                                                                             Base Peak
                                                                                                                                                      MS
                                  17.79
                                 388.10
                                                    118.32
                                                    431.94 140.05             258.97
                                                                                                                       240 min                        a431_240mi
                      50                                               228.84 776.40                                                                  ng_1029
                                                           1839.77     619.48        269.22
                                                                                     444.83
                      0                                                                                                                               NL:
                                                                                                                 432.24            520.91
                                                                                                                                                      4.60E8
          100
                                8.40
                               439.73
                                                                                                                 675.17480 min
                                                                                                                             1912.57
                                                                                                                                                      Base Peak
                                                                                             341.64 362.07 382.40                      524.05         MS
                                                                                             675.16 675.25 675.26       465.75
                                                                                                                                       1511.36        a431_1213_
                                                               171.60 226.50 268.84 294.93                              488.12
                      50        12.29         113.04 123.05
                                                     926.00    1154.56 794.78 1285.77 576.29                                                 575.63
                                                                                                                                                      8hrg
                                390.90        897.74                                                                                         444.75
                      0
                           0            50       100      150          200     250       300       350      400      450         500        550   600
                                                                                      Time (min)
2D LC-MS/MS of proteins from A431
cell lysates
 Analysis of proteins from A431
           cell lysates
Gradient   # of Proteins
             Identified    Total Run Time   # of Proteins
  1D
                                 2D           Identified
 30min          16
                                5 hr.           144
 60min          22
                               10 hr.           337
120min          44
                               20 hr.           491
240min          56
480min         105
                                Yeast Protein separation
RT: 0.00 - 147.01
                                                                    43.98                                                                               NL:
              100                                                                                                                                       1.37E9

                      80              13.19
                                                   25.63     43.10                                 20 mM Ammonium chloride,                             Base Peak
                                                                                                                                                        MS
                      60                                                                                                                                Yeast_120mi
                                                                                                                                                        nG_01
                      40                                                45.49
                               3.85                                      48.38
                      20                                                    53.42                                                     130.22
                                                                                        62.96     79.48      97.29 111.60   121.87
                0                                                                                                                                       NL:
                                                    27.67          43.08
              100                                                                                                                                       4.29E9
 Relative Abundance




                      80                      23.30                                                 40 mM Ammonium chloride,                            Base Peak
                                                                                                                                                        MS
                      60                19.68                39.16                                                                                      yeast_120mi
                                                                                                                                                        ng_02
                      40                               29.34
                                    18.10                                  51.05                                                         132.90
                                                                                   53.67                               116.91   128.66
                      20         14.07
                                                                                     61.26      75.82   94.59 104.07                           135.25
                0                                                                                                                                       NL:
                                                    28.23
              100                        24.11                                                                                                          2.67E9

                      80
                                                                                                    70 mM Ammonium chloride,                            Base Peak
                                         23.21             33.86    36.15                                                                               MS
                      60                                                                                                                                yeast_120mi
                                                                    44.14                                                                               ng_03
                                                                               57.31
                      40               16.50
                                  14.65
                      20                                                            62.00
                                 10.12                                                          75.84      97.14 102.90          122.37 130.00
                0                                                                                                                                       NL:
                                                            38.40 46.50       57.21
              100                                  29.91                                            100 mM Ammonium chloride,                           4.13E9
                                                                                                                                                        Base Peak
                      80                                                                                                                                MS
                      60                                                                                                                                yeast_120mi
                                              25.02                                                                                                     ng_04
                      40
                                  15.27 19.79
                      20                                                             58.33                                      124.47   132.44
                               1.30
                                                                                                75.95     98.84 101.21
                       0
                           0                  20                   40              60              80            100            120               140
                                                                                         Tim e (m in)
                               Yeast Protein Separation
RT: 0.00 - 147.01
                                                       32.77            52.66
                                                                                           140 mM Ammonium chloride,                                 NL:
              100                                                                                                                                    4.05E9
                                                                                                                                                     Base Peak
                      80                                                                                                                             MS
                                                                                                                                                     yeast_120mi
                                                                                                                                                     ng_05
                      60                  18.84                 46.16       57.14
                                                            44.10
                      40
                                      12.57
                      20
                               1.57                                             58.03      75.86
                                                                                                      87.56   96.28       121.87 129.57 137.22
                0                                                                                                                                    NL:
                                                               41.06
              100                                                                                                                                    3.24E9
                                                                                            180 mM Ammonium chloride,                                Base Peak
 Relative Abundance




                      80                                                                                                                             MS
                                                                                                                                                     yeast_120mi
                                                  26.85                                                                                              ng_06
                      60
                                              22.22
                      40               13.34
                                                       32.88
                                                                       50.93
                      20        12.54                                                                                              128.39
                                                                           54.17     57.88 75.96                                            132.41
                                2.55                                                                  87.63   89.93       124.39
                0                                                                                                                                    NL:
                                          18.52
              100                17.01                                                                                                               1.02E9
                                                                                            220 mM Ammonium chloride,                                Base Peak
                      80                                                                                                                             MS
                                                                                                                                                     yeast_120mi
                                                                                                                                                     ng_07
                      60
                                                               42.07
                               1.34                 27.73
                      40       1.89                    30.91
                                                                                           75.86
                                                                       50.18 57.70
                      20          14.82                                                        76.99
                                                                                       70.04            87.67
                                                                                                                97.96 113.09 129.14    134.87
                       0
                           0                   20              40               60               80             100         120              140
                                                                                       Tim e (m in)
                                         Yeast proteins
Reference                                                                    Score Hits          Entries
                                                                           65809.5 18 4
gi|129922|sp|P14828|PGK_KLULA PHOSPHOGLYCERATE KINASE gi|66895|pir||KIVKGL 4 3 0                 91 95 96 101 255 260 686 689 824 873 888 902 913 925 926 94
gi|6319594|ref|NP_009676.1| translational elongation factor EF-1 alpha; Te 56564.0 12 1 1 0 0    76 215 239 336 1020 1024 1159 1163 1338 1362 1518 1526 : 1
                                                                           46500.4 gi|1
gi|119145|sp|P16017|EF1A_CANAL ELONGATION FACTOR 1-ALPHA (EF-1-ALPHA) 0 11 1 1 0                 : 76 215 239 1020 1024 1159 1163 1338 1362 1518 1526 : 113
                                                                           46303.5 12 1
gi|1172457|sp|P41757|PGK_CANMA PHOSPHOGLYCERATE KINASE gi|83923|pir||JT095 0 0 0                 255 260 824 860 873 888 913 925 926 948 960 1050 : 994 : : :
gi|6324637|ref|NP_014706.1| Ribosomal protein L3 (rp1) (YL1); Rpl3p [Sacch 42592.4 10 0 1 1 0    133 138 143 383 387 468 471 482 484 1531 : : 386 : 1305 :
gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae] gi|1 41268.3 10 2 1 0 0    38 56 58 69 77 262 269 270 302 313 : 180 1413 : 163 : :
gi|6319279|ref|NP_009362.1| Pyruvate kinase; Cdc19p [Saccharomyces cerevis 38052.7 23 3 0 0 0    29 38 59 70 73 92 132 136 166 288 291 421 474 483 900 1121
gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae] gi|1 36687.9 13 2 1 0 1    8 11 39 42 129 377 396 486 488 590 802 1751 1814 : 1014 162
gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae] gi|1 34389.6 13 2 1 0 1    19 50 329 349 441 442 445 820 1028 1037 1434 1522 1548 : 34
gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 34030.4 13 7 0 0 0    49 113 116 212 215 336 567 608 804 1082 1275 1317 1320 : 12
gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase; Pgk1p [Saccharomyc 26362.2 7 4 1 0 0     185 187 307 313 404 1743 1785 : 109 373 1746 1804 : 1811 : :
gi|6322790|ref|NP_012863.1| aldolase; Fba1p [Saccharomyces cerevisiae] gi| 26245.4 2 0 0 0 0     266 269 : : : :
gi|6319279|ref|NP_009362.1| Pyruvate kinase; Cdc19p [Saccharomyces cerevis 25546.9 9 0 0 0 0     61 131 218 276 309 310 1005 1399 1404 : : : :
gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 23954.0 7 1 0 0 1     97 143 204 294 356 365 492 : 297 : : : 264
gi|6325112|ref|NP_015180.1| sequence similar to Hes1p; Kes1p [Saccharomyce 23937.7 3 0 2 2 0     611 736 1234 : : 76 1362 : 215 1547 :
gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase; Pgk1p [Saccharomyc 23524.5 3 2 1 0 1     171 173 298 : 175 1060 : 1226 : : 378
gi|6321609|ref|NP_011686.1| phosphatidylserine decarboxylase located in va 23254.1 2 0 1 3 4     475 1103 : : 404 : 397 451 1007 : 139 185 400 1607
gi|6322526|ref|NP_012600.1| phosphatidylinositol kinase homolog; Tor1p [Sa 21082.5 0 3 2 1 2     : 7 80 770 : 1518 1690 : 1024 : 75 1020
gi|14318479|ref|NP_116614.1| Actin; Act1p [Saccharomyces cerevisiae] gi|1121011.4 2 0 0 0 0      372 384 : : : :
                                                                           20063.6 IN
gi|12230852|sp|O74343|YH2X_SCHPO HYPOTHETICAL 76.4 KDA PROTEIN C1A4.09 0 1 1 1 1                 : 1603 : 847 : 1607 : 1590
                                                                           DEHYDRATASE)
gi|119336|sp|P00924|ENO1_YEAST ENOLASE 1 (2-PHOSPHOGLYCERATE 19580.1 4 0 0 0 0                   177 260 302 313 : : : :
gi|6324313|ref|NP_014383.1| Pbi2p [Saccharomyces cerevisiae] gi|124818|sp|19318.9 5 0 0 0 0      62 70 73 1878 1882 : : : :
gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 19151.9 7 0 0 0 1     208 211 213 215 1467 1609 1610 : : : : 1288
gi|6325331|ref|NP_015399.1| Transketolase 1; Tkl1p [Saccharomyces cerevisi 18932.7 5 1 0 0 0     217 218 223 224 1328 : 420 : : :
gi|6321631|ref|NP_011708.1| Glyceraldehyde-3-phosphate dehydrogenase 3; Td 18650.3 3 0 0 0 0     1590 1603 1607 : : : :
gi|6321977|ref|NP_012053.1| 6-phosphogluconate dehydrogenase; probable GND 18647.0 4 0 0 1 0     647 1529 1530 1534 : : : 695 :
gi|7492246|pir||T40586 nucleolar protein involved in pre-rRNA processing - 18417.0 6 8 12 10 8   1075 1243 1276 1414 1508 1540 : 1009 1076 1247 1482 1585 1
gi|7491534|pir||T40003 hypothetical protein SPBC25H2.08c - fission yeast   18270.9 0 1 2 0 0     : 143 : 133 138 : :
gi|6325016|ref|NP_015084.1| 82 kDa heat shock protein; homolog of mammalia 18036.9 6 2 0 0 0     5 31 132 800 1229 1235 : 37 1237 : : :
              Yeast proteins
gi|6323376|ref|NP_013448.1| Ribosomal protein L26A (L33A) (YL33); Rpl26ap 4748.4 2 1 1 0 0    1624 1627 : 1625 : 652 : :
gi|6321007|ref|NP_011086.1| Transcriptional regulator which functions in m 4747.8 0 0 0 1 0   : : : 33 :
gi|6323292|ref|NP_013364.1| Ylr262c-ap [Saccharomyces cerevisiae] gi|21318 4746.0 0 1 0 0 1   : 876 : : : 732
gi|6322314|ref|NP_012388.1| Yjl147cp [Saccharomyces cerevisiae] gi|1353019 4744.7 0 0 0 1 0   : : : 460 :
gi|6324034|ref|NP_014104.1| Ynl295wp [Saccharomyces cerevisiae] gi|1353106 4744.4 0 1 0 0 0   : 396 : : :
gi|6324151|ref|NP_014221.1| Ribosomal protein S3 (rp13) (YS3); Rps3p [Sacc 4743.3 2 0 0 0 0   279 332 : : : :
gi|6323691|ref|NP_013762.1|                                                4741.6 0 0 3 0 3   : : 1877 1949 2145 : : 981 1314 1853
gi|6322806|ref|NP_012879.1| p58 polypeptide of DNA primase; Pri2p [Sacchar 4738.8 0 0 1 0 1   : : 971 : : 558
                                                                           4737.8 0 0 0 1
gi|1171671|sp|Q09711|NCS1_SCHPO HYPOTHETICAL CALCIUM-BINDING PROTEIN C18B1 0                  : : : 397 :
gi|7496455|pir||T19442 hypothetical protein C25A1.7a - Caenorhabditis eleg 4737.0 0 0 1 2 1   : : 1597 : 185 1645 : 1521
gi|83218|pir||S19440 hypothetical protein YCR029c - yeast (Saccharomyces c 4736.0 0 0 1 0 0   : : 50 : :
gi|6323487|ref|NP_013559.1| Ylr454wp [Saccharomyces cerevisiae] gi|13637324735.9 1 1 2 2 0    724 : 6 : 638 1305 : 359 1200 :
gi|6319642|ref|NP_009724.1|                                                4735.1 0 0 0 0 1   : : : : 224
gi|6319850|ref|NP_009931.1| non-mitochondrial citrate synthase; Cit2p [Sac 4729.1 1 1 0 0 1   646 : 1020 : : : 1619
gi|6324552|ref|NP_014621.1| 3'-5' exoribonuclease complex subunit; Dis3p [ 4728.2 0 0 1 1 0   : : 1637 : 329 :
gi|7490292|pir||T38695 conserved hypothetical protein SPAC3C7.09 - fission 4726.5 0 0 1 1 0   : : 766 : 1441 :
gi|6320126|ref|NP_010206.1|                                                4724.8 0 0 0 2 3   : : : 342 712 : 598 637 1671
gi|1077218|pir||S49776 hypothetical protein YDR179w-a - yeast (Saccharomyc 4723.5 0 0 1 0 0   : : 50 : :
gi|1352297|sp|P48996|DP27_CAEEL CHROMOSOME CONDENSATION PROTEIN DPY-27 1 2 4723.3 1 0 0 gi|   1196 : : : 482 : 122 896
gi|6323875|ref|NP_013946.1| involved in silencing; Esc1p [Saccharomyces ce 4722.7 1 0 1 0 1   1103 : : 1722 : : 792
                                                                           4719.0 IN
gi|1175464|sp|Q09796|YAA2_SCHPO HYPOTHETICAL 111.5 KD PROTEIN C22G7.02 1 0 1 0 1              1954 : : 294 : : 214
gi|6323762|ref|NP_013833.1| Ymr115wp [Saccharomyces cerevisiae] gi|2497155 4717.5 0 0 0 1 0   : : : 243 :
gi|6322171|ref|NP_012246.1| Ribosomal protein L2B (L5B) (rp8) (YL6); Rpl2b 4715.9 1 1 0 0 0   42 : 25 : : :
gi|2495228|sp|Q12578|HIS7_CANGA IMIDAZOLEGLYCEROL-PHOSPHATE DEHYDRATASE (I 4715.9 0 1 0 0 0   : 610 : : :
                                                                           4715.5
gi|15213983|sp|O94489|EF3_SCHPO ELONGATION FACTOR 3 (EF-3) gi|7492556|pir| 1 0 1 0 0          24 : : 326 : :
gi|6321594|ref|NP_011671.1| Cystathionine beta-synthase; Cys4p [Saccharomy4712.9 2 0 0 0 0    284 652 : : : :
gi|6323722|ref|NP_013793.1| (putative) involved in sister chromosome cohes 4712.4 1 0 0 0 1
                                                                            (METHYLAMIN
gi|113719|sp|P12807|AMO_PICAN PEROXISOMAL COPPER AMINE OXIDASE4712.2 4 6 4 3 5
                                                                                              Protein # 1708
                                                                                              561 : : : : 198
                                                                                              1168 1335 1362 1468 : 1169 1228 1471 1496 1499 1613
gi|6320232|ref|NP_010312.1|                                                4707.7 0 0 0 0 1   : : : : 202
gi|6322088|ref|NP_012163.1|                                                4707.5 0 0 0 2 0   : : : 116 1740 :
2D LC-MS/MS of Yeast proteins

     • Time: 15 hours

     • Gradient: 5 – 65% Acetonitrile in
       2 hrs in each step

     • Proteins searched by
       Bioworks 3.1

     • Proteins identified: 1708

     • Throughput: 113.8 proteins/hr
Viewing Results
           TIC




Synclein
 alpha
Filters for SEQUEST Results

 Xcorr:+1>1.5, +2>2.0, +3>2.5
 ∆CN: >0.1
 When three or fewer peptides for an
  individual protein passed the criteria
 (1) the spectrum quality (S/N, match rate)
 (2) some continuity must be present among
  the b or y fragments
 (3) if proline is predicted to be present, then
  the corresponding y fragment should give an
  intense peak.
 (4) unidentified intense peaks should be
  verified as being either doubly charged.
Filters for SEQUEST Results
On-Line Phosphopeptide
Enrichment (IMAC capture)
Flow Path of an Automated 2D (IMAC + RP)-MS/MS System
for the Analysis of Phosphopeptides
                                 1D-IMAC Column

                                                            10 –port valve
                                                            in mass
            Injector                                        spectrometer
  Sample loop
                                          IMAC
                        2
                                          SCX


                                  1
                                                  RP 1
                                                 Column 1

            3         Pump 1
                Sample valve
                      injector        6                                      LCQ Deca XP
                  4
                            5                                                Plus- mass
                                                                             spectrometer
                                                            To Waste
                               Sample Pump


                                          Analytical Pump
   Procedure Used for Automated 2D LC(IMAC+RP)-
   MS/MS Analysis of Phosphopeptides

Step 1: Load IMAC column

Step 2: Load peptides on IMAC column.
      Flow-through peptides captured by RP2 column.

Step 3: Wash IMAC column. The bound peptides are then
eluted by phosphate buffer on to RP1, while the flow-
through peptides trapped on RP2 are being analyzed by
LC/MS.

Step 4: The bound phosphopeptides on RP1 are analyzed by
      LC/MS/MS.
Capture of FQ*SEEQQQTEDELQDK Phosphopeptide of -Casein Digest in
the 2D LC(IMAC+RP)-MS/MS System

                      Non-phosphorylated peptides
                      flow through IMAC column and           NL: 1.34E9
  RP2 column          captured by and eluted from RP2

                    position for m/z=1031.7 on C2 column




                       Phosphorylated peptide (m/z=1031.7,
   RP1 column          FQ*SEEQQQTEDELQDK)                    NL: 1.80E8
                       captured by IMAC column, bound to
                       RP1, and eluted.
Neutral Loss Scanning Confirmed the Major
Ion at m/z=1031.6 as a P-peptide




                      Neutral loss
                      fragment (-49)


         MS/MS of 1031.6          M+2H+-49

                                   Phosphorylated peptide (m/z=1031.7,
                                   FQ*SEEQQQTEDELQDK)
Bioworks 3.1 Search Identified the P-peptide
with m/z=1031.6 as FQ*SEEQQQTEDELQDK
               (M+2H)-49              (M+2H)-49
         1+                     2+
Proteins - Differential
Expression
 (EGF treated and untreated
cells)

     ----Alternative method for differential
 Protein differential expression
1. Divide A431 cell sample in two:
 a) Half stimulated by EGF
 b) Half control
2. Lyse cells
3. Extract proteins from lysates
4. Digest with trypsin
5. Run 2D LC-MS/MS of digests with
   ProteomeX
6. Proteins identified by TurboSEQUEST
   software
7. Compare “stimulated” vs. “control”
             Automated 2D LC-MS/MS Analysis of
             Human A431 Cell Proteins
RT: 0.00 - 87.01                                                                                                                                    RT: 0.00 - 87.01
                                                                                                                               NL:                                                                       35.16                                                         NL:
                                                     36.47                                                                                                                                                                50.21
                        11.84                                                                                                                                                                                                                                          9.00E8
  100             8.17 652.2       20.80
                                  1154.5 27.76
                                                     974.2
                                                                                  4 NH Cl 0 mM
                                                                                                                               4.02E9
                                                                                                                               Base Peak              100                           18.73         30.76  947.6            991.8                            60 mM       Base Peak
                  513.5                                                                                                        MS
                                                                                                                                                                                    893.3 19.50 832.0                                                                  MS
                            14.06                                                                                                                                            14.64        654.6                                                                        a431_04040
                6.03                       866.2 34.75                                                                         A431_04040
                            981.2                                                                                              2_01                                          653.5                                            51.82                                    2_05
               652.9                             814.4                                                                                                  50             8.92                    27.91
    50                                                                                                                                                                                                                        522.2
                                                         37.28 45.66                                                                                                   578.0                   653.8          35.81 41.74
                                                         974.1 955.3 51.00 53.93 59.86 66.46 74.00 77.03                                                                                                     1286.3 322.0       53.03 59.38 64.81 72.10 80.53 83.25
                                                                     614.2 804.3 1179.0 971.0 1010.4 1024.2                                                                                                                     522.6 842.3 371.0 390.8 370.9 370.9
     0                                                                                                                                                   0                                                                                                             NL:
                                  20.34            34.70 37.53                                                  NL:                                                                       23.93
                                  896.2            970.7 974.2                                    10 mM         7.25E8                                                                    797.2
                                                                                                                                                                                                                                                          80 mM
                                                                                                                                                                                                                                                                       1.10E9




                                                                                                                               Relative Abundance
  100                                       31.70                                                               Base Peak                             100                                            31.93                                                             Base Peak
                                      26.50 925.6          38.01     50.68                                      MS                                                                                   866.3                                                             MS
                          13.92                                                                                 a431_04040                                                                                                                                             a431_04040
                                      784.4               1335.0     496.2                                                                                                                    26.10
                    10.95 581.5                                                                                 2_02                                                             16.37        878.6                       50.62                                        2_06
    50                                                                                                                                                  50                12.80 600.1
                    566.9                                                                                                                                        1.22                                                     496.2
             1.21                                           42.02        52.10 54.90                                                                                      495.8                               38.72 42.18
                                                                                     65.23    73.06 78.78 85.51                                                  712.5                                                             56.97 65.29 69.94 73.18 78.99 85.04
             712.6                                          322.0        522.3 716.3                                                                                                                         1103.9 322.0
                                                                                     369.3    494.5 371.0 371.0                                                                                                                    315.9 369.3 508.7 508.6 370.9 370.9
     0                                                                                                                         NL:                       0
                                         23.83                                                                                                                                                                   33.02                                                           NL:
                                                                            50.28
  100
                                        1154.7
                                                                            991.6                20 mM                         6.96E8
                                                                                                                               Base Peak              100                                      24.01 27.32
                                                                                                                                                                                                                 932.8
                                                                                                                                                                                                                                     51.72              120 mM
                                                                                                                                                                                                                                                                                 3.95E8
                                                                                                                                                                                                                                                                                 Base Peak
                                                                                                                               MS
                                                                                                                                                                             11.49             697.0 889.5                     50.30 522.3                                       MS
                                   22.25                                                                                       a431_04040                                                                                      496.2                                             a431_04040
                                                                                                                                                           1.35              417.3
                                   820.7           31.16 37.57                                                                 2_03                                                                                                                                              2_07
    50                     20.32                  1113.3 974.6
                                                                                51.75                                                                   50 712.5                                                     36.50
                                                                                                                                                                                                                           38.62
             1.36                                                    46.23      522.2                                                                                    9.30                                        900.1
                     13.92 896.1                                                                                                                                                                                           894.5
             712.6                                                   955.2              54.11      68.69 74.10         85.00                                             630.8                                                            52.55 59.49 69.19 72.16 80.72 83.45
                     464.4
                                                                                        760.2     1418.3 1010.4        370.9                                                                                                              522.4 842.4 1269.7 390.9 371.0 370.9
     0                                                                                                                         NL:                       0
                              18.07                                                                                                                                                            22.56                                                                             NL:
                                                                                                                               1.26E9
  100
                              652.3                                                              40 mM                         Base Peak              100
                                                                                                                                                                                               682.7
                                                                                                                                                                                                                                                        160 mM
                                                                                                                                                                                                                                                                                 1.04E9
                                                                                                                                                                                                                                                                                 Base Peak
                                        23.73                                                                                  MS                                                                          30.16
                                                                                                                                                                                                                                                                                 MS
                                        839.4                                                                                  a431_04040                                                                  822.8                                                                 a431_04040
                                                  28.85                                                                        2_04                                                    17.43
    50                                                                        52.37                                                                                              15.47             23.90                           50.63 52.17                                   2_08
                         14.08                   1079.1 36.47                                                                                           50                             480.8
             1.06                                                            1043.7                                                                              0.82            478.7             668.8         32.76             496.3 522.2
                                                        814.4       45.61
             712.6 12.34 655.0                                      955.3
                                                                                        56.98 62.62    72.50 81.03 84.35                                         712.4                                           932.6 38.09 42.17             56.86 72.03 73.04 80.31 86.81
                   729.2                                                                315.9 563.3    390.9 370.9 370.9                                                                                               894.7 321.9
     0                                                                                                                                                                                                                                         315.9 508.5 508.7 370.9 370.9
                                                                                                                                                         0
         0            10           20              30           40         50              60         70          80
                                                                                                                                                             0               10            20               30            40         50         60         70         80
                                                                Time (min)
                                                                                                                                                                                                                          Time (min)
Automated 2D-LC-LC/MS-MS Analysis of Human
A431 Cell Proteins (continued)
                         RT: 0.00 - 87.02
                                                        15.05                                                                                                         NL:
                                                                                                                                                                      4.11E8
                                                        506.3                                                     51.86
                           100                                                                                                                                        Base Peak
                                                                                                                  522.4                                               MS
                                      1.20                                                                 50.26                                                      A431_04040

                             50
                                      712.5
                                                                                                 41.68
                                                                                                           496.3                      200mM                           2_09
                                                13.02                         31.88
                                          9.31 503.1       15.60                                 322.1
                                                                        29.42 569.4                                         59.03 62.99     72.08         82.29
                                          452.8            473.0
                                                                        677.9                                               886.5 347.9     391.0         370.8
                              0                                                                                                                                       NL:
                                      1.14                                                                      52.16                                                 2.46E8
                                      712.6                                                               50.64 522.2
                           100
    Relative Abundance




                                                                                                          496.2                                                       Base Peak
                                                                                                                                                                      MS
                                                                                 32.45            42.15                                                               a431_04040
                                                                                 569.6            321.9                                                               2_10
                             50        1.91       12.85
                                                  603.4     19.02                                         49.37                       300mM
                                       712.5                               30.87     32.95                                         63.71        77.53 79.64 85.51
                                                            857.9                    932.5                496.5       53.47
                                                                           832.1
                                                                                                                      508.3
                                                                                                                                   347.9        370.9 370.9 370.9
                              0                                                                                                                                       NL:
                                      1.09                                                                    50.36
                                                                                                                                                                      2.95E8
                                      712.4                                                                   991.7
                           100                                                                                                                                        Base Peak
                                                                                                                    51.73                                             MS
                                                                                                                   1043.5                                             a431_04040
                                                                                                                                                                      2_11
                             50        2.00                                      32.17
                                                                                                 41.92                                500mM
                                       712.5 9.27 15.78                          569.7
                                                                        25.96                    321.9                                                 81.51
                                             446.4 824.2                                                                  59.23 63.24      72.13 78.98
                                                                        769.1                                                                          370.9
                                                                                                                          886.4 348.0      390.9 370.9
                              0                                                                                                                                       NL:
                                      1.04
                                                                                                                                                                      2.70E8
                                      712.5
                           100                                                                                                                                        Base Peak
                                                                                                              50.64                                                   MS
                                        1.69                                     32.37                        496.3                                                   a431_04040
                                                                                                  42.27               52.17
                                        712.4
                                                5.69                             569.6
                                                                                                  321.9               522.3
                                                                                                                                      900mM                           2_12
                             50                                 19.62                                                                      70.87
                                                712.5                                    37.95
                                                                712.5      29.70                          46.88                    63.72   508.7 72.54        83.46
                                                                                         331.9                        54.66
                                                                           347.9                          399.1                    348.0         390.9        370.9
                                                                                                                      371.1
                              0
                                  0             10               20             30               40         50                60           70            80
                                                                                                 Time (min)



   Total Proteins Identified= 709, using Bioworks 3.1 with TurboSequest
   (Xcorr = 1.5, 2.0, and 3 for charge states +1, +2, and +3, respectively )
Proteins Differentially Expressed in Control and EGF-
Stimulated A431 Cells
Proteins Differentially Expressed in Control and EGF-Stimulated
A431 Cells (continued)




  *Only those proteins with two or more
  peptides identified were compared
Proteins Identified in Both Control and
EGF-Treated A431 Cells
Proteins Common to Control and EGF-treated
A431 Cells (continued)
Proteins Common to Control and EGF-
treated A431 Cells (continued)
  Differential Protein
      quantitation
-quantitative proteomics
Stable isotope labeling (SIL)
 for quanlitative proteomics

Metabolic labeling (13C,   15N)


Post-biosynthetic labeling (ICAT
 reagent)

Post-digest isotope Labeling of
 tryptic peptides(18O)
Metabolic labeling with [13C6]Arg in
 the elucidation of EGF signaling
 Cells were grown in medium containing either
  normal or [13C6] arginine.
 8 h of serum starvation, the labeled cells were
  stimulated with 150ng/ml EGF for 10 min, whereas
  the unlabeled cells were left untreated.
 Cells were lysed and combined in a 1:1 ratio
  followed by incubating at 4°C with Grb2 fusion
  protein bound to GSH-sepharose beads for 4h.
 Wash with lysis buffer, boiled in sample buffer,
  and resolved on a 4-12% gel.
 Bands of interest were excised and subjected to in
  gel digestion.
 Mass spectrometric analysis
                      SH2 domain of Gb2 binds
                      tyrosine-phosphorylated
                      proteins including EGFR, Shc
                      etc.,




Strategy to study activated EGFR
            complex
Quantification of protein ratios from peptide doublets. Top panels show
mass spectra of peptides of different identified proteins, bottom panels
show mass spectra of peptides from EGF-stimulated cells upon detection of
      Metabolic labeling
Advantages: (1) all sample-to-sample
 variability induced by subsequent
 biochemical experiments can be
 eliminated. (2) metabolism-related
 dynamic labeling involved in a
 specific physiological process.
 Drawbacks: (1)only works in cell
  culture systems that tolerate
  isotope-substituted media (which is
  actually often not the case), which
  may not be compatible with a
  particular biological investigation.
  (2)Total isotope substitution is
  required for reliable for MS-based
  quantification, which renders the
  approach rather expensive.
  (3)Difficulty in establishing an
  enrichment method
     Differential Quantitation with isotope-coded
     affinity tags (ICAT)
1.    Divide the previous sample (hGH in plasma) into two identical pools.

2.    Reduce and alkylate (D0 ICAT for one plasma pool and D8 ICAT for the other),
      separately. Mix the two pools and digest the whole mixture with trypsin.
                                                 ProteomeX (2D)
3.    Sample clean-up
      a) ion exchange to remove excess ICAT reagent
      b) avidin affinity to capture the ICAT-labeled peptides
                                       Collect the flow through Frxn   ProteomeX(2D)
4.    Collect the ICAT-peptide fractions and run LC-MS/MS.
                                                ProteomeX (1D)
5.    Data analysis by Bioworks 3.1
      a) TurboSEQUEST for protein identification
      b) XPRESS for relative quantitation
The structure of ICAT reagent
Data Dependent Mass Tag Setting for
              ICAT


                                 1+
                                 2+
                                 3+
  TurboSEQUEST Search Parameters




Turbosequest parameters are set as usual except the amino acid modification and differential mass need
to be set as in above
    Bioworks 3.1 (SEQUEST and XPRESS)
200mM NH4Cl   Search Results
Differential Quantitation by Bioworks (XPRESS)
                    Software

                                                               *
                                                         NYGLLYCFR
                                                         (T16 peptide of human growth hormone)




 After finishing the TurboSEQUEST search, click the XPRESS function to locate the correct cysteine-
 containing peptide sequence (identified from its MS/MS spectrum) with the ratio of D0 and D8 ion
 intensities (integrated from its parent ion spectrum) as shown in above.
RT: 35.09 - 35.37
                                                    Zoom In MS spectra
                                                                                                                             NL:
                                                                                                 35.29                       9.68E5

       100
                              (+2) Charge D8              M/Z = 799.1 Signal = 0.986                                         m/z=

                      90                                                                                                     MS
                                                                                        35.25
                      80                                                                                                     Base Peak

                      70                                                                                     35.33
                                                                35.20                                                        798.6-799.6
 Relative Abundance




                      60

                      50
                                                35.16
                      40

                      30

                      20

                      10                          35.16

          0
       100                                                                                                                   NL:
                      90    (+2) Charge D0                 M/Z = 794.9 Signal =1.09                          35.33
                                                                                                                             1.09E6
                                                                                                                             Base Peak
                      80
                      50
                      70
                                                                               D0/D8 = 1.1                                   794.4-795.4
                                                                                                                             MS
                      40
                      60                                                    Using the highest MS intensity
                      30                                        35.20                                                        m/z=
                      20

                      10
                                                                                                35.29
                       0
                           35.10        35.15                 35.20                  35.25         35.30             35.35
                                                                        Time (min)
Advantage: (1)Largely reduce the
 complexity of peptide mixture;
 (2)Easy to enrich.
Drawbacks: (1) 14% protein sequences
 do not contain cysteine-containing
 tryptic peptides (800-2500Da),19%
 contains just a single such peptide
 (alternatively, cleavable ICAT
 reagents). (2) requirement of protein
 over 100 mg.
Post-digestion isotope labeling
             ( 18O)

 Artifacts (i.e. side reactions)
  inherent to chemical labeling can be
  avoided.

 All peptides can be used for
  identification and quantification

 Available for gel-separated proteins
 Samples of interest are first digested with
 trypsin.

 Aliquots are subsequently incubated with
 either   16O   water or18O water in the presence of
 trypsin. Labeling efficiencies of individual
 peptides of the H218O-treated sample are
 determined by MALDI-TOFMS of a small portion
 of the sample. Mixtures of     16O-   and   18O-labeled


 samples are then applied on the MALDI plate,
 and relative abundances are derived from
General scheme of post-digest   18O   labeling
                procedure
Time course of trypsin-catalyzed post-digest labeling of 1 pmol BSA tryptic
digest. The exchange rate of C-terminal oxygen atoms is dependent on the
peptide sequence. Fast exchanging peptides show complete labeling after <10
min (a). However, for some peptides close to quantitative labeling could only
be achieved after incubation for 2 h (c).
Practical considerations for
 stable isotope labeling in
  quantitative proteomics
Predictable mass difference
  between labeled and unlabeled
  samples

Easy to enrich
An example of Data dependent MS/MS mode-
reject high abundant proteins(GDH-2)
Glutamate dehydrogenase 2




               1193.29




  1759.92
Data dependent setup for rejecting
high abundant GDH-2      Just ion of
                           interest
Post-Translational
   Modifications
Modifications
Modifications (continued)
Modifications (continued)
Phosphorylatio
       n
Protein identification:
     Phosphorylation
Data Dependent (with Dynamic Exclusion)
      MS/MS spectrum of m/z 980-982
                                                     Y”12+1 Y”10+1
                                Arg-Leu-Ser-Leu-Val-Pro-Asp-Ser-Glu-Gln-Gly-Glu-Ala-Ile-Leu-Pro-Arg

                         100

                          90                                                      Serine
                                                                              Phosphorylated
  % Relative Abundance




                          80      Serine Not
                                Phosphorylated
                          70                           931.8 (MH2 - H3PO4)2+
                          60

                          50

                          40
                                                                                       Y’’12+1
                          30                                                           1311.5

                          20
                                                                       Y’’10+1
                                                            922.7
                                                                       1099.5
                          10                        764.6
                                   452.5551.1                                               1410.6   1591.5
                                                                                                          1689.7
                               366.4          665.3                  1083.1                                    1786.8
                          0
                                 400        600       800           1000        1200        1400      1600      1800
Glycosylations
Glycosylation
Glycosylation
Glycosylation
Identifying Glycosylation – MS full scan
   RT: 0.00 - 140.00
                                                                                             46.78                                                                                                                       NL:
                   100
                                                                                                                                                                                                                         2.79E10
                           90
                                        Glycopeptide region                                                                                                                                                              TIC MS

                           80
                                                                       31.08
                           70
                                              MS from                          34.55                           Other region perform only MS and MS/MS
                                                             26.55 29.80                44.66
      Relative Abundance




                           60
                                      5.32           1
                           50                                                                                56.23
                                                      2
                           40
                                         6.06                3                                                                                                    97.61
                           30
                                                              4                                      49.19                                                    96.92
                                             8.59                                                                57.42                                       95.93    98.25            113.39   121.72
                           20                                21.91
                                                     21.55                                                           61.02                           88.24                    110.98        117.35
                                                    13.23                                                                     64.91
                                                                                                                                      70.50 75.92 86.75
                           10                                                                                                                                                                        122.46     138.88
                            0
                                0             10          20           30              40            50              60         70             80        90           100          110         120        130
                                                                                                                             Time (min)


                                                                       # 583      RT: 15.57           AV: 1 NL: 7.23E8
   T: + c Full ms [ 200.00-2000.00]
                                 527.5
                                                                                 Glycopeptide region                                                                                                     Fu
            20                                                                                                                                                        N
                                                                                        MS scan                                                                                                               As
                                                                                                                                   glycopeptide ion
   Relative Abundance




            15
                                    217.0
                                                                                                                                                                                                              n
                                                                      575.8                                                                                                (select to do MS/MS)
            10
                                                                                                                                      +3
                           5         234.1                                                                                    1064.0
                                                       445.0
                                          286.8                             634.8           762.3     807.9               1060.7      1095.7        1267.6
                                                                                                                                                                                         +2
                                                                                                                                                                          1514.8   1595.2            1780.2              1987.9
                           0
                               200                  400                    600                  800                       1000                 1200              1400                  1600              1800              2000
                                                                                                                                      m/z
Identifying Glycosylation – MS/MS
                                      584 RT: 15.59
   T: + c Full ms2 1064.00@65.00 [ 280.00-2000.00]
                                                                                  :                             +2
                                                                                                             1267.6
          100                                                                                                                                    Fu
                         95                                                                                                                           Asn
                         90

                         85
                         80                                                                                                    (select to do MS to 3)
                         75

                         70

                         65

                         60
    Relative Abundance




                         55

                         50
                         45                                                                         +2                               +2
                         40                                                                               Fu
                                                                                                                Asn N                 Fu
                         35                                                                                                                Asn
                         30                                                                         1185.8                                            +2
                                                                                                                          1413.1
                         25
                                                                                                                                                      Fu
                                                                                                                                 1449.6                    Asn
                         20
                                                                                      +2                                 Fu
                                                                                                                               Asn
                                +1                     +1                               Fu                            1369.4
                         15                                                                  Asn
                                                N                                                                                    1450.3
                         10   366.0
                                                    657.0                            1003.5
                          5                                 739.5                              1085.1                                1478.9
                                      453.8                               923.0   966.8                                                                          1845.7   1933.3
                          0
                                 400          600                   800               1000               1200             1400                1600          1800              2000
                                                                                                   m/z
Identifying Glycosylation – MS3
  TPA_iontree_2_010524173638      # 585 RT: 15.61 AV: 1 NL: 4.09E5
  T: + c Full ms3 1064.00@65.00 1267.64@65.00 [ 335.00-2000.00]
                                                                                                             1333.2
         100
                       95
                                                                                                                                     As      (select to do MS to 4)
                       90
                       85
                                                                                                                                     n
                       80
                                                                                 oxidized
                                                  +2                                     Fu
                       75                   Fu                                                Asn
                                                                                                                                 Fu
                       70
                                                    As                          1011.3                                                Asn
                       65
                       60
                                                    n
                                                  666.6                                                                     1478.3
  Relative Abundance




                       55                                                                                                       1479.2
                                                                             993.5
                                                                                                Fu
                       50                                                                         As
                       45                                                                         n                                   Fu
                       40
                                                          +2                                              Fu Asn
                                                                                                                                           Asn
                                                          Fu                           1085.2                                   1537.6
                       35                                                                       1185.7             1334.6
                                                               Asn
                       30                              740.3
                                                                     892.4
                                                                                                                                                                        1987.5
                       25
                                  528.0
                       20                                                                                                                                    Fu
                                     551.2                 768.6                                                                                                  Asn
                       15                                                                                                                    1697.2
                                          586.1                                                                       1460.4
                       10                                               930.3                            1315.8                                          1859.1
                        5
                        0
                            400           600                  800              1000             1200               1400              1600            1800               2000
                                                                                                m/z
Identifying Glycosylation – MS4
  TPA_iontree_2_010524173638        # 586 RT: 15.63 AV: 1 NL: 8.63E4
  T: + c Full ms4 1064.00@65.00 1267.64@65.00 1333.22@65.00 [ 355.00-2000.00]
                                       728.4                                  1213.4
    100
                       95                        B6                                           Dehydro-alanine form
                       90                        (select to do MS to 5)                       Further CNH2 loss on N-terminal     B6
                       85
                                                                                                                                B5      B7
                       80
                       75
                                                                                                               C-T-S-Q-H-L-L-N-R
                                                                     Peptide only
                       70                                                                                      SCH2COOH                 y
                       65
                                                                 C-T-S-Q-H-L-L-N-R
                                                                                1130.7                                                  2
                       60                                                                                           Y7
  Relative Abundance




                       55
                       50
                                  506.2
                       45
                       40
                       35                                                  Y7                         Dehydro-alanine form
                                                 B7      833.0             -H2O                   C-T-S-Q-H-L-L-N-R
                       30                                              1053.4
                       25             B5 636.2   -H2O
                                                 823.4                                       1240.5
                       20   Y2 / Y6(+2)
                              492.2
                                      618.3      798.0

                       15
                       10
                        5
                        0
                            400           600      800              1000              1200              1400         1600        1800        2000
                                                                                     m/z
Summary of one glycopeptide fragmentation
    pathway (a biantennary glycopeptide)

          MS                                  MS/MS                    MS to 3
                                                            Fu               Fu
   N                 Fu
                                                                 Asn                Asn
                          Asn                                              (1267 +2)
                                                                              Fu
                                                                                       Asn
       211                                    210                           (1186 +2)
       (1064.4 +3)                            (1450.6 +2)
                                                                                 Fu
                                         LCQ-deca (nanospray)                          Asn
                                                                            (1085 +2)

                                                                                 Fu
                                                                                       Asn
                                                                             (1105 +2)

                                                                                 Fu
           LCQdecaXP (microspray)                                                      Asn
                                                                             (1004 +2)
           MS to 4 or 5
                          CTSQHLLNR(1333 +1)                                           Asn
                                Peptide only (1131 +1)                             (1333 +1)
De Novo Peptide Sequencing
Why De Novo Peptide Sequencing ?

    Determination and/or confirmation of
    peptide sequences derived from
    proteins that are:

    not in the databases (including
     DNA sequence)
    with amino acid modifications
 De novo sequencing software
  (PARSER II)

 Ref: Zhang ZQ, McElvain JS.
  De Novo peptide sequencing
  by two-dimensional fragment
  correlation mass spectrometry.
  Anal Chem, 2000, 72 (11):
  2337-2350
MS, MS2 and MS3 spectra collected with
             peak parking
    100                                                                                               496.1

    80                       Base Peak
    60
                                                                                                               Full Scan MS
    40
    20
                                                                                    730.4                                    990.6
    0                                                                                *
                16      18   20     22      24         100
                         Time (min)                                                                  400      600      800         1000     1200
                                                                                                                       m/z
                                                       80     Full Scan MS2
                                                       60                                                               713.4
          120.2
                                                                  389.2
                                                       40    261.1 *                                                         Full Scan MS3
                 Full Scan MS3                            233.1 *
                                                             *
                                                                          616.4
                                                                     502.4 *                                                 @730.4
                                                       20
                 @233.1                                                       732.4
                                                        0    200    400 600     800           1000
                                                                        m/z                                   389.2    714.5
         86.1                                                                                                    502.3
                121.2
                                                                                                               400          800           1200
           100       200     300      400                                                                                    m/z
                           m/z

                                                                                                                    599.3
                  233.1                                              354.1                                                  Full Scan MS3
                                                                 261.2
                             Full Scan MS3                   226.1         Full Scan         MS3                            @616.4
                             @261.1                                           @389.2
                                                                            372.2
                                                                    243.0                                       581.2

                                                            129.1
                            234.1                                                                      200 400 600 800 1000
            0 100                                                   200      400       600                      m/z
                          200   300      400     500
                                m/z                                           m/z
Determination of Peptide Sequence by MS3 De Novo
  Sequencing Software --- Biowork 3.1

                           Peptide = FINNIGANK
Sequencing Tryptic Peptide (m/z 585.1)
  by MS3 De Novo Sequencing Software


                    Peptide = TGPNLHGLFGR
Thank You!

				
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