Deducing protein composition from complex protein preparations by MALDI without peptide separation.
.
TP #419
Kenneth C. Parker SimulTof Corporation, Sudbury , MA
Introduction
How best to characterize a proteome of intermediate complexity from
a large number of distinct samples?
Investigate the following:
1.) Digest with trypsin, and collect MALDI spectra.
2.) Collect msms spectra from the mixture.
11
18
13
56
80
81
1 MYH1
2 ACTA1
3 MYL3
4 CKM
5 ACTN2
6 MYH2
7 ALDOC
8 MYLPF
9 PK
10 MYL1
11 CKS
12 PGK
13 HSPB
14 GAPDH
15 ENO
16 TPM
1
1
1
1
1
1
1
2
Table 4. MSMS searches
31
27
56
43
70
65
83
84
96
76
78
86
85
100
105
110
120
Figure 2.
Two overlapping precursors at mz 1869.94 (#76):
This precursor mapped to myosin, but was off by
37 ppm. Two prominent components are visible at
higher resolution; one component is very close.
Only 2 of 12 putative correct IDs that are off by >
10 ppm cannot be so readily explained. 37
peptides matched to < 10 ppm even from low
resolution parent spectra.
How to tell correct from incorrect MS-MS IDs without cheating ?
(but rules deduced by cheating)?
1.
2.
3.
Length Protein Name
Pyruvate Kinase
Myosin light chain 1, cardiac muscle
Creatine kinase S-type
Phosphoglycerate kinase
Heat shock protein beta-1
Glyceraldehyde-3-phosphate DH
Enolase
Tropomyosin alpha
82
86
Table 1. Summary of PMF protein level results
Peptide Count
Score
%CM %IM ppw
All
PMF(u) PMF (t) msms
1941 Myosin heavy chain fast isoform 3
423
59
61 15
360759 35.9 20.1 8.0
377 Actin, alpha skeletal muscle
56
12
12 9
135126 36.0 16.7 3.6
150 Myosin light chain 3
38
7
7 4
36041 47.9 5.3 3.6
381 Creatine kinase M-type
64
11
14 3
31889 35.1 2.8 4.4
897 Alpha-actinin-2
174
20
29 1
11944 19.0 4.4 12.4
761 Myosin heavy chain
198
15
34 3
6449 18.2 5.8 15.8
772 Glucocorticoid receptor
111
12
13
4816 20.8 2.3 10.9
180 Uncharacterized protein
27
5
6
2266 32.7 1.4 4.9
362 Fructose Bisphosphate aldolase mouse
75
5
8 1
1787 19.3 1.1 12.1
205 UPF0454 protein C12orf49 homolog
37
4
5
889 22.2 1.2 9.3
152 Acetylcholinesterase
18
3
3
875 20.8 0.8 4.3
212 Fibroblast growth factor 10
40
7
8
626 26.6 1.8 13.8
157 Calcimedin, 32K
23
3
4
429 22.7 0.5 4.4
498 Nuclear factor 1
57
5
5
319 21.0 1.0 13.5
168 Myosin regulatory light chain 2
32
3
6 2
304 31.9 0.3 8.6
10
47
62
60
79
24
40
31
1.
2.
3.
4.
5.
6.
Symbol
Green-> correct by PMF
Blue-> Correct by MSMS but not PMF
#
Methods
I
Table 3. Peptide level comparison
8
6
1
What are the limitations?
1. Dynamic range / protein complexity
2. Overlapping peptides / incomplete precursor isolation.
3. Incomplete database
Buy chicken meat from grocery store.
Mince, solubilize, reduce, alkylate, precipitate, trypsin digest.
Dilute into matrix and collect MALDI spectra.
Database used : chicken NCBI with 15148 sequences.
Perform PMF on the digest (using proprietary software).
Collect 120 MS-MS spectra in triplicate on precursors between mz
700-3200, and consider up to 1 missed cleavage.
7. Compare results with PMF and to previous data collected following the
standard HPLC-MSMS approach.
8. All identifications considered to be false if protein is not known to be
abundant.
9. How many correct IDs at the protein and peptide level can be
obtained?
10. What explains false identifications?
Rank
.
Figure 1. The below parent spectrum should be color-coded according to success of msms. Green-> all 3 correct, Blue-> 2 correct; yellow-1 correct, red-> incorrect, grey-> no id Data
on individual spectra are listed in Table 4.
amu peptides peaks I
1
269
68
1
269
68
1
269
68
1
269
68
1
269
68
1
2
3
4
5
Pep MZ ppm Sequence
TrS
Symb
1869.87 27.3 aSSDAEMAIFGEAAPYLR
1995 MYHsk_k
1870.08 -83.4 DLLLQTRLINDVTSIR
758 RyR3
1869.97 -27.1 DLIELQALIDSHFEAR
642 TNNT3
1869.98 -31.1 DLYALFEQILEKTMR
591 MYO9A
1869.91
3.7 SQYVPYDGIPFVNAGSR
562 ALS2CR8
1
1
1
1
1
269
269
269
269
269
29
29
29
29
29
1
2
3
4
5
1869.87
1869.97
1869.91
1870.08
1869.95
27.3 aSSDAEMAIFGEAAPYLR
-27.1 DLIELQALIDSHFEAR
3.2 YHAIVYPMKFMQGER
-83.4 DLLLQTRLINDVTSIR
-15.1 SSEVETTVFYIPGVDVK
1748 MYHsk_k
976 TNNT3
817 NPSR1
774 RyR3
617 KIAA1109
5
5
5
5
5
1384
1384
1384
1384
1384
29
29
29
29
29
1
2
3
4
5
1869.87 27.3 aSSDAEMAIFGEAAPYLR
1869.97 -27.1 DLIELQALIDSHFEAR
1867.87 1093.9 SIGEDVYEKPMSELDR
1869.91
3.2 YHAIVYPMKFMQGER
1868.84 579.1 DNEQLSHLNQEQEER
1748 MYHsk_k
976 TNNT3
893 FILIP1
817 NPSR1
816 GOLGA2
20
20
20
20
20
5675
5675
5675
5675
5675
29
29
29
29
29
1
2
3
4
5
1869.87 27.3 aSSDAEMAIFGEAAPYLR
1869.97 -27.1 DLIELQALIDSHFEAR
1867.87 1093.9 SIGEDVYEKPMSELDR
1869.91
3.2 YHAIVYPMKFMQGER
1868.84 579.1 DNEQLSHLNQEQEER
1748 MYHsk_k
976 TNNT3
893 FILIP1
817 NPSR1
816 GOLGA2
4.
5.
6.
7.
High mass accuracy. Mass accuracy becomes an important filter (20 ppm filters out 2/3 of
1 amu wrong IDs) even when the resolution is between 5K and 10 K as in Figure 1.
Complexity of the parent mass region. Much stronger parent masses 10 amu away may
interfere.
Consistency of identification between replicate MSMS spectra (Only 3 wrong but consistent
IDs with score > 805 found)
Robustness of ID as a function of peak detection.
Robustness of ID as a function of precursor mass accuracy.
Difference in score between top hit and highest incorrect hit (Table 2)
Longer peptides are more reliable. Short homologous wrong peptides often get high
scores. See Table 5.
<-Table 2
From this msms spectrum, the program correctly identifies myosin, starting with either 29 or 65
peaks detected, when searched against the chicken database considering a 1 amu search
window, a 5 amu window, or a 20 amu window. With the shorter peak list (with less confounding
noise), the 2nd hit is a troponin peptide, known from HPLC MSMS to be abundant in chicken
muscle preparations. It may explain the 2nd parent mass visible in the high resolution spectrum.
Table 5. Hits to spectrum #1
PepMW mz
ppm Sequence Score PIM PCM Symbol
774.40 774.32 104.1 EEIDIR
3360 84
56
774.40 774.32 104.1 EEVEIR
3360 84
56 C9orf64
774.45 774.32 175.3 EILMIR
2835 70
63
774.35 774.32 34.1 &DIDIR
2386 79
47 ACTA1
774.40 774.32 104.1 EIEDLR
2280 70
57
774.40 774.32 105.3 qMIMIR
2131 65
51 CUL4B
774.45 774.32 165.7 EQTKLR
2099 71
46
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
msms
774.32
779.37
795.45
917.47
942.46
945.55
948.47
976.44
992.52
1000.49
1010.52
1028.54
1045.53
1053.54
1060.53
1093.55
1107.63
1130.56
1153.57
1156.66
1170.55
1173.55
1188.66
1192.60
1200.69
1217.62
1233.62
1243.66
1253.61
1269.59
1296.66
1314.71
1338.66
1342.69
1359.68
1373.73
1382.67
1386.74
1391.76
1398.76
1401.76
1421.72
1473.78
1483.69
1488.73
1501.71
1512.70
1515.74
1521.73
1532.78
1540.73
1553.75
1556.81
1559.83
1566.78
1605.76
1609.78
1611.78
1618.80
1657.84
1660.81
1702.83
1724.85
1730.91
1749.78
1765.89
1768.98
1772.89
1778.93
1790.89
1803.88
1812.93
1816.91
1825.00
1854.89
1869.94
1885.89
1896.90
1924.94
1956.05
1974.92
1986.90
1992.96
2013.91
2070.92
2199.05
2215.05
2246.02
2256.05
2262.05
2277.06
2289.09
2298.11
2307.09
2313.11
2320.11
2329.13
2336.07
2365.10
2374.09
2386.10
2393.11
2425.12
2436.11
2448.19
2460.13
2464.12
2478.12
2494.14
2499.12
2539.17
2563.20
2634.19
2689.12
2704.29
2777.15
2784.15
2838.19
3067.58
3100.39
ppm
180.1
21.1
25.4
-0.4
58.6
1.2
10.4
10.6
-33.0
0.2
8.9
17.1
36.8
-37.3
-8.4
-26.9
0.1
-10.7
61.7
-5.7
86.5
-3.8
-39.8
-3.1
-59.4
-2.5
-21.3
-8.4
4.4
-22.3
-7.7
15.3
57.7
4.2
-18.1
10.1
12.2
-63.9
-29.0
2.5
-65.1
32.7
-5.8
-5.8
-17.5
-18.0
-1.0
-5.0
8.4
9.7
6.3
-3.6
0.3
-21.9
-92.6
-19.7
3.0
-7.1
-33.1
-1.1
16.7
0.1
70.1
-4.4
4.8
28.6
-45.2
0.2
35.7
0.9
-12.7
1.1
6.7
-33.0
43.5
-37.4
13.3
2.5
45.2
-4.8
2.2
1.6
0.6
-0.5
-8.2
0.7
-0.1
-1.2
53.1
-30.8
58.0
-10.3
17.4
69.6
-66.1
-3.3
26.8
8.4
45.3
129.9
37.4
44.4
40.3
42.5
1.8
70.3
10.2
-19.4
102.9
67.2
66.6
-3.4
100.2
101.5
40.9
97.4
88.4
89.3
-1.8
-302.2
Correct; Score > 805
Wrong; Score > 805 sc
< 10 ppm > 805 10 ppm <20 ppm Total Distinct
0
3
3
2
2
3
3
1
1
1
0
1
1
1
3
1
3
3
3
3
0
2
2
1
3
1
3
1
1
1
3
3
1
2
2
3
3
3
1
2
1
1
1
1
3
1
3
1 1*
3
1
2
1
1
1
1
1
0
0
3
1
3
3
1
1
1
2
2
0
1
1
1
0
2
1
2
1
1
1
0
0
1
3
1
1
1
1
1
3
2
3 3*
3
1
1
2
1
0
3
1
3
1
2
1
3
1
1
3
2
1
3
1
1
3
2
1
1
1
1
1
3
3
3
1
2
1
1
1
3
3
3
1
3
1
2
1
3
2
1
1
1
1
1
1
0
0
0
0
1
1
2
1
1
1
2
1
2
2
1
3
1
1
1
3
1
1
1
1
1
1
1
1
1
3
3
1
3
1
1
0
1
3
37
116
15
2
2
PepMW Seq
3103
1418
2215
1144
1207
5006
1195
2989
1540
5208
3733
920
1109
650
2040
649
1239
1980
1284
1903
1170
801
925
2415
1137
4935
1180
1918
566
741
1861
949
875
801
732
826
910
1296
947
2263
1696
707
825
3974
1269
1228
6715
660
850
913
736
2056
1178
984
897
1615
1368
4983
441
1142
1155
4401
623
2363
2320
515
508
494
664
5839
1012
1008
838
481
555
2391
658
2536
486
4680
5406
2325
840
1650
547
3208
1882
5666
672
572
640
650
499
763
536
2214
650
741
632
675
584
799
432
466
1244
330
519
431
526
447
734
829
580
408
956
633
673
600
1968
722
774.46
779.39
795.47
917.47
942.52
945.55
948.48
976.45
992.48
1000.49
1010.53
1028.55
1045.56
1053.50
1060.52
1093.52
1107.63
1130.55
1153.64
1156.66
1170.65
1173.55
1188.62
1192.60
1200.62
1217.62
1233.59
1243.65
1253.62
1269.56
1296.65
1314.73
1338.73
1342.70
1359.66
1373.74
1382.69
1386.65
1391.72
1398.76
1401.66
1421.77
1473.77
1483.68
1488.71
1501.68
1512.70
1515.73
1521.74
1532.80
1540.74
1553.75
1556.81
1559.79
1566.64
1605.73
1609.78
1611.77
1618.75
1657.83
1660.83
1702.83
1724.97
1730.90
1749.79
1765.94
1768.90
1772.89
1778.99
1790.89
1803.85
1812.93
1816.92
1824.94
1854.97
1869.87
1885.91
1896.91
1925.02
1956.04
1974.93
1986.90
1992.96
2013.91
2070.90
2199.05
2215.05
2246.02
2256.17
2261.98
2277.19
2289.07
2298.15
2307.25
2312.96
2320.10
2329.19
2336.09
2365.20
2374.40
2386.19
2393.22
2425.22
2436.21
2448.19
2460.30
2464.15
2478.07
2494.39
2499.28
2539.34
2563.19
2634.45
2689.40
2704.40
2777.42
2784.39
2838.44
3067.57
3099.45
TGTARIR
ALzYPR
IIAPPER
ELSNGLER
AIGHFELR
AVFPSIVGR
MLQIzAGR
AGFAGDDAPR
ETLVSQzR
&NGVLEGIR
EAFLLFDR
TIPWLENR
SSEGVQGILR
MNYIEDLR
NASQTRNNR
GIEEDEFVR
VPPPLPQFGR
GYSFVTTAER
IVLPDVMNPR
LVIIEGDLER
ELTPQVVSAAR
GYSLPPHzSR
QPMSATLRER
EAFTVIDQNR
ASPEVEQTALR
DLFDPVIQDR
EGNGTVMGAELR
IQLVEEELDR
DSIMVQEYIR
ELTYQSEEDR
DTQIHLDDALR
TVVPARzGVTVR
eFGFLATRLFR
NGASALPRHTYR
MLHAYNPSRDR
YTLPPGVEATAVR
LGESQLSGPAHMR
DPLEIPDSGSGGSR
ADALTEEINFLR
ANLLQAEIEELR
IDQASLDYSYAR
TQzLQYLRALR
qAFTQQIEELKR
qEYDEAGPSIVHR
IEELEEEIEAER
DTGTYEDFVEGLR
DQGTFEDFVEGLR
GDHTNRDGIIFDR
TSSTFEPSPKPSEK
GTFTPVVTDPITER
QTPTREYVDFER
VVIGMDVAASEFzR
VPTPNVSVVDLTzR
ETTADPSELEAIKR
zSPETSLMPDzNR
qPAHDAWAEDVDLR
SFLTAASPGEKGSSDR
SFLEELLTTQzDR
PHMQASPQQPNNNR
TFLVWVNEEDHLR
AGMEKTSAITAAAADPR
LQNEVEDLMIDVER
LAAGFLLLFQFLSER
TPGAMEHELVLHQLR
LVSWYDNEFGYSNR
qPLRPPzVLAFVTER
HIPGSPFTAKITDDNR
DILSPDMISPPLGDFR
ITPVSPVHHPPLSELR
SYELPDGQVITIGNER
&QENADPQKMAFLLR
AzANPAnGSVILLENLR
DISDGFPPGDATIVTRR
SAGVHQPKFAAEIAENR
EGDRIIQInGIEVQNR
aSSDAEMAIFGEAAPYLR
ETEGFVARDFTMLVDR
HADSTAELGEQIDNLQR
GENLAIGFDIHKVELNR
VAPEEHPTLLTEAPLNPK
YPIEhGIITNWDDMEK
MEGDLNEMEIQLSHANR
GTGGVDTAAVGAVFDISNADR
MEGDLNEMEIQLNHANR
FzGWYDADLSPAGQQEAR
NDLQLQVQAEADALADAEER
NDLQLQVQAEADSLADAEER
DLYANNVMSGGTTMYPGIADR
IDDVPPIKFELSQGSPQMVR
SSSSEzSLKEDLQTzMFPR
GGSLLzGTSAAKAVLTLSTQADR
DLTPLSDGDVGSSTSSHSSQRR
DEMAHVTGRVLPAPMLQYGGR
NPNSLKAMAPLSSGVNLPLLDR
SSLTPSSQDDSSNQEDGQESSK
LETDIAQIQSEMEDTIQEAR
qAASTPALSPAGPAASPPGAGSGKAAR
LETDISQIQSEMEDTIQEAR
DADVFPIEVDLARTTLNYGQK
FGLPVIPPTIPFQRGLAPLNVK
QPPSVPnGPSSPITESAPELPQR
eEAADSPELSPALEELIQKVSR
ASPSAIGSSQPPSVTPASSPSKEQK
GPPGPVGPSGKDGSNGMPGPIGPPGPR
KLETDIAQIQSEMEDTIQEAR
YHYTSSSLPRNLPINITNTIR
GEDGDPGQPGPPGPSGEAGPPGPPGKR
DINEzALDPDIzSNGIzENLR
SPFPVTVAPPLQLDKVSVQGLSSK
VEVLPPIETKGLTSDDVSDLTDR
EVPPSPALSTGTVLPNADLTYQLR
qEAPPHIFSISDNAYQFMLTDR
PEVPSHVPFLLIGGGTAAFAAARSIR
TPLHTNELNDIQSQPGGYHLLTLK
qPPPPARPAAALGPDPGVPGAPMGFSLR
MPILEHPAYPPATLLzLESLVPzR
ePPRYMAPFSVVSNLSTIHIDWSR
GALGQPGPAGEQGMRGPQGPPGQIGTPGIR
TVPPAVPGVTFLSGGQSEEEASLNLNAINR
PEPPPAQGPALALzHYSDLSNNNDFYVL
Acc
MYHsk_k
ACT
ACT
CENPM
ACT
MYHsk_k
MYL1sk
ACTN1
ZCCHC11
CKMT2
ACTA1
ZBTB43
TPM1
VCL
CKM
C10orf78
MYLPF
RHBDF2
CKM
MYL_K
TPM1
JAK
MYH3
MYHsk_k
BRAF
KIAA0922
HSPB1
RERE
HOXA2
MYH3
MYH3
ACTA1
MYHsk_k
MYL_K
MYL1sk
MPRIP
ENO3
GAPDH
LD
FLCN
PKM2
MYLPF
CKM
DCLK2
MYH3
PLXDC2
MYHsk_k
GAPDH
NPNT
CDC42EP3
ACT
CENPL
PGK
LAO
PTPRJ
PDZRN3
MYHsk_k2
PEX1
MYHsk_k
CAPN5
ACTA1
ACTA1
MYHsk_k
CKM
MYH2f_k
PGAM1
MYH2f_k
MYHsk_k
ACTA1
KIAA0922
KIAA1244
C6
EIF2C3
EPHB2
RPS6KC1
MYH2f_k
cDtx2
MYH_N127
PICK1
RAR-gamma2
CAPRIN2
COL2A1
MYH2f_k
AGPAT2
CD1.1
MYHsk_k
OBSCN
LVRN
USH2A
COL6A3
ALDOC
Int
73
86
62
41
82
7
83
21
11
29
81
89
16
52
12
58
70
87
68
25
28
31
67
38
48
65
77
100
47
75
99
35
54
57
94
24
13
34
98
26
39
6
56
43
22
2
79
49
59
23
61
69
97
74
46
80
5
93
19
45
96
44
17
9
55
15
72
3
32
78
1
37
88
91
64
95
53
10
40
27
30
66
63
92
20
18
8
60
33
51
84
85
14
50
42
76
4
36
71
90
Mass
211
196
240
328
202
997
200
525
800
437
207
186
744
278
792
260
215
192
220
495
443
432
224
354
286
228
209
173
290
211
175
414
270
261
182
500
780
416
175
456
332
1209
264
315
509
2723
207
285
255
507
243
220
177
211
304
207
1483
184
587
304
180
304
656
873
265
756
213
2539
430
208
7311
391
192
185
228
182
275
850
331
446
437
227
234
185
547
598
925
244
423
279
200
199
775
284
317
210
2090
401
214
185
747.39
774.32
779.37
795.45
917.47
945.55
948.47
976.44
1000.49
1010.52
1028.54
1045.53
1060.53
1107.63
1130.56
1156.66
1171.59
1174.60
1192.60
1200.69
1201.68
1217.62
1233.62
1243.66
1296.66
1314.71
1316.65
1338.66
1342.69
1359.68
1377.67
1382.67
1385.76
1386.74
1390.70
1391.76
1398.76
1401.76
1412.76
1421.72
1473.78
1483.69
1487.74
1488.73
1501.71
1512.70
1515.74
1516.75
1532.78
1553.75
1556.81
1559.83
1567.79
1570.89
1605.76
1609.78
1611.78
1618.80
1657.84
1660.81
1663.82
1701.84
1702.83
1730.91
1744.86
1749.78
1768.98
1772.89
1778.93
1781.91
1790.89
1795.90
1803.88
1810.95
1816.91
1825.00
1854.89
1869.94
1885.89
1890.86
1896.90
1902.91
1924.94
1936.93
1956.05
1974.92
1986.90
1992.96
2013.91
2070.92
2115.12
2131.12
2199.05
2215.05
2228.04
2244.08
2246.02
2320.11
2322.09
3067.58
ProtR
symbol
1
1 MYHsk_k
10 ACTA1
1
2 ACTA1
1
2 ACTA1
1 MYHsk_k
3 MYL1sk
5 ACTN1
28
1 MYHsk_k
33 CKMT2
2
1
2
5
7 MYLPF
3
1
4
3
5 TPM1
1 MYHsk_k
7
12
14
7
6
19
1
5
27
7
8
1 MYH3
6
6
5
1 MYH3
2 ACTA1
14
1 MYHsk_k
12 MYL_K
3 MYL1sk
2
34
1
17 ENO3
11 GAPDH
1
34
29
28 PKM2
16
2 MYLPF
1
4 CKM
1
13
27
1 MYH3
1 MYHsk_k
29
32 GAPDH
38
6
1
25
2 ACTA1
24
9
23
1
8
31
22 MYHsk_k2
28
21
1 MYHsk_k
45
1
14
2 ACTA1
2 ACTA1
1 MYHsk_k
4 CKM
38 MYH2f_k
31
28
35
28 MYH2f_k
1 MYHsk_k
25
10
2 ACTA1
1 MYH2f_k
35
9 ALDOC
ppm
Sequence
-16.4 SEIDRK
msms
-21.1 ALzYPR
6.2 QHLLER
0.5 AALEQTER
-1.2 AVFPSIVGR
14.3 TEELEEAK
-10.6 AGFAGDDAPR
-0.2 &NGVLEGIR
-8.9 EAFLLFDR
-17.1 TIPWLENR
-23.5 ADAPVKWMK
15.2 ANSEVAQWR
13.8 LNNNIKYTK
10.7 GYSFVTTAER
-13.6 NLRNTQAVLK
15.7 HQGVMVGMGQK
-7.6 EGLLLWzQR
-15.5 IMSTLNMLGGR
-1.2 ITLSQVGDIVR
15.5 AGLLGLLEEMR
2.5 DLFDPVIQDR
21.3 EGNGTVMGAELR
16.5 HRPDLIDYSK
7.7 DTQIHLDDALR
7.3 INDRGDLLPSSK
-15.8 SYNHLQGDVRK
-23.6 RSLPSTSSTSSTK
-12.3 HMLMVARELSR
-10.7 GRLQTENGELSR
-19.9 TLFFRYMSQGK
9.4 IQHELEEAEER
-24.6 LASELLEWIRR
10.5 LDTTHHPRQPGK
6.6 zLQAGMNLEARK
2.8 GDAVTEIGITFLR
-2.5 ANLLQAEIEELR
23.9 LAEQELLEATER
-8.9 NNLLLAEVEELR
11.0 GYEEWLLNEIR
5.8 qAFTQQIEELKR
5.8 qEYDEAGPSIVHR
-4.8 AFAYTWFNLQAR
17.5 IEELEEEIEAER
-16.9 HnGRQMFVALnGR
1.0 DQGTFEDFVEGLR
-7.7 IWHHTFYNELR
-7.2 SQKTPTSSPSPGSQK
-9.7 LLSSIDVDhTQYK
9.7 GQNHNVQGFHPYR
-8.0 GIASYLNVWYSRK
-1.5 IVESLQSSLDAEIR
17.1 EQDTDSINKQVYK
17.3 QVPPQFLETVEKR
-20.9 DFzSAQLSVKEPPK
18.6 &PFAHGEFPDPKPK
2.2 qEYDEAGPSIVHRK
18.4 LEEAGGATAAQIDMNK
1.1 TFLVWVNEEDHLR
-8.0 NAYEESLDHLQTLK
-1.6 GMGTDEEEILILTSR
-9.4 SQDGEELAISGGGGLRR
-0.1 LQNEVEDLMIDVER
4.4 TPGAMEHELVLHQLR
-6.5 QWQMNIEGVNDVALK
-12.9 RSISzPSzNGLAEGNK
-6.6 PILQRYISLLMEHR
-18.5 DLQMRLDEAEQIALK
-6.7 DLQHRLDEAEQLALK
-2.6 KDVDGAYMTKVDLQAK
-0.9 SYELPDGQVITIGNER
9.7 DVFLGmFLYEYARR
-14.1 &PLPRPWALTFSYGR
16.1 VPFTFLTSPSWEPFR
1.7 RQAEEAEELSNVNLSK
1.3 ELFVLTYGALVAQLzK
16.7 QALLHzQDFHDQSQK
6.7 SADLTAMSDNKELYLAK
11.8 GYIVEMQDEGSTDWKK
-1.1 YGSGGGSKGGSLSGGGYGSGGG
-2.5 HADSTAELGEQIDNLQR
6.3 KQDEYHMVHLVzTSR
12.2 YETDAIQRTEELEEAK
-17.9 VSASHHAPDRPPDPFPPL
4.8 VAPEEHPTLLTEAPLNPK
-2.2 YPIEhGIITNWDDMEK
-1.6 MEGDLNEMEIQLSHANR
-0.6 GTGGVDTAAVGAVFDISNADR
-9.6 SQLDSRNSSSSSSLASSEGK
-24.4 LIQEQYLQTESzSSAGEK
-1.1 IIAEAEISDTPLLTSEEKR
4.6 ASIQLPMEKAIETALDzLK
-17.2 zSITTTEKSzILEILDSTK
0.1 NDLQLQVQAEADSLADAEER
2.5 LDSELKNMQDMVEDYRNK
-10.1 FLNRAAMAFQNLFMAVEDR
1.2 DLYANNVMSGGTTMYPGIADR
3.3 LETDIAQIQSEMEDTIQEAR
3.7 FLzNMTFLKEYMEEEIPK
1.8 TVPPAVPGVTFLSGGQSEEEASL
ALzYPR
IIAPPER
AVFPSIVGR
AGFAGDDAPR
&NGVLEGIR
EAFLLFDR
TIPWLENR
ANSEVAQWR
VPPPLPQFGR
GYSFVTTAER
EAFTVIDQNR
ITLSQVGDIVR
DLFDPVIQDR
EGNGTVMGAELR
IQLVEEELDR
DTQIHLDDALR
ANLLQAEIEELR
qAFTQQIEELKR
qEYDEAGPSIVHR
IEELEEEIEAER
DTGTYEDFVEGLR
DQGTFEDFVEGLR
VVIGMDVAASEFzR
VPTPNVSVVDLTzR
qPAHDAWAEDVDLR
SFLEELLTTQzDR
TFLVWVNEEDHLR
LQNEVEDLMIDVER
TPGAMEHELVLHQLR
LVSWYDNEFGYSNR
SYELPDGQVITIGNER
aSSDAEMAIFGEAAPYLR
HADSTAELGEQIDNLQR
VAPEEHPTLLTEAPLNPK
YPIEhGIITNWDDMEK
MEGDLNEMEIQLSHANR
GTGGVDTAAVGAVFDIS
MEGDLNEMEIQLNHANR
NDLQLQVQAEADALADAEER
NDLQLQVQAEADSLADA
DLYANNVMSGGTTMYP
LETDIAQIQSEMEDTIQEA
TVPPAVPGVTFLSGGQSE
49
Conclusions:
4th hit obviously correct
•Using PMF, can identify most abundant proteins
•Samples like chicken muscle, plasma, whole blood lysate, saliva have a rather small
number of abundant proteins that can be compared by the means shown here.
• Confirmation of PMF by MSMS can be performed directly on unseparated digests,
resulting in ~15 protein IDs from whole chicken muscle.
•Failures usually due to weak precursors close to strong precursors, or mixtures.
•Similar analyses have been successfully performed on human biofluids ,
(not shown)