Introduction to 2D LCMS/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
Attomole Sensitivity !!!
New Inter-Octapole Lens
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
Spot 1
100
Found t-PA
50
22.49
13.98
10.92
28.29
74.38
51.42
42.27
66.11
0
100
2
13.77
50
0
100
19.53
21.95
3
3.68
51.90
28.64
17.20
65.73
45.20
Found t-PA
51.85
23.16
22.05
41.22
35.66
70.49
82.75
82.59
97.56
83.90
100.31
82.29
95.10
61.76
100
4
50
0.48
8.82
0
100
21.83
23.06
5
50
4.34
15.18
34.16
20.55
29.58
17.44
36.77
43.07
51.89
65.77
51.72
59.20
0
80.86
82.44
100.61
65.88
79.87
82.27
97.30
61.60
100
6
50
1.26
12.70
0
100
7
50
8.54
11.13
21.90
19.62
27.77
37.34
22.74
21.28
48.81
51.85
70.37
51.83
29.56
44.47
6
8
48.00
63.79
54.56
70.37
72.64
81.26
79.86
81.19
85.70
84.87
98.74
92.89
0
0
2
4
10
Time (min)
12
14
16
NL: 1.41E7
Base Peak F: + c Full ms [
300.00-2000.00] MS
gelspot_tpa4_c2
NL: 2.11E7
Base Peak F: + c Full ms [
300.00-2000.00] MS
gelspot_tpa5_c1
63.93
Found t-PA
39.32
35.12
42.52
70.29
NL: 9.02E6
Base Peak F: + c Full ms [
300.00-2000.00] MS
gelspot_tpa2_c2
NL: 1.16E7
Base Peak F: + c Full ms [
300.00-2000.00] MS
gelspot_tpa3_c1
79.67
72.35
70.58
40.14
0
Relative Abundance
75.40
61.66
22.68
50
NL: 2.39E7
Base Peak F: + c Full ms [
300.00-2000.00] MS
GelSpot_tPA1_C1
69.67
18
20
NL: 1.15E7
Base Peak F: + c Full ms [
300.00-2000.00] MS
gelspot_tpa6_c2
NL: 8.00E6
Base Peak F: + c Full ms [
300.00-2000.00] MS
gelspot_tpa7_c1
Global Protein Identification
Global Protein
Identification
SCX column fractionation
Protein mixture
Reverse column
separation
Protein digests
Auto MS/MS detection
Results
BioWorks data base search
Tandem MS spectra
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 LCMS/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
431.84
100
0
26.66
652.24
42.92
1138.32
100
Relative Abundance
50
0
50
100
NL:
2.65E9
Base Peak
MS
a431_60min
g_1029
60 min
76.43
667.61
68.71
50.66 1160.53
486.92
100
0
30 min gradient
35.12
1163.80
50
NL:
2.28E9
Base Peak
MS
A431_30min
G_1029
117.86
563.10 138.63
703.32
NL:
1.28E9
Base Peak
MS
a431_120mi
ng_1029
120 min
148.78
371.00
14.27
344.05
17.79
388.10
50
0
84.72
200.37
1154.55
563.16
118.32
258.97
431.94 140.05
228.84 776.40
1839.77
269.22
619.48
444.83
50
240 min
432.24
675.17
341.64 362.07 382.40
465.75
675.16 675.25 675.26
171.60 226.50 268.84 294.93
488.12
1154.56 794.78 1285.77 576.29
520.91
480 min1912.57
8.40
439.73
100
12.29
390.90
113.04 123.05
926.00
897.74
524.05
1511.36
575.63
444.75
0
0
NL:
1.49E9
Base Peak
MS
a431_240mi
ng_1029
50
100
150
200
250
300
Time (min)
350
400
450
500
550
NL:
4.60E8
Base Peak
MS
a431_1213_
8hrg
600
2D LC-MS/MS of proteins from A431
cell lysates
Analysis of proteins from A431
cell lysates
Gradient
1D
# of Proteins
Identified
30min
16
60min
22
120min
44
240min
56
480min
105
Total Run Time
2D
# of Proteins
Identified
5 hr.
144
10 hr.
337
20 hr.
491
Yeast Protein separation
RT: 0.00 - 147.01
NL:
1.37E9
Base Peak
MS
Yeast_120mi
nG_01
43.98
100
20 mM Ammonium chloride,
43.10
25.63
13.19
80
60
45.49
48.38
53.42
40
3.85
20
Relative Abundance
0
100
27.67
62.96
121.87
130.22
NL:
4.29E9
Base Peak
MS
yeast_120mi
ng_02
40 mM Ammonium chloride,
19.68
60
97.29 111.60
43.08
23.30
80
79.48
39.16
29.34
40
18.10
14.07
20
0
100
51.05
53.67
61.26
75.82
94.59 104.07
116.91
128.66
132.90
135.25
NL:
2.67E9
Base Peak
MS
yeast_120mi
ng_03
28.23
24.11
80
33.86
23.21
60
44.14
16.50
14.65
10.12
40
20
0
100
70 mM Ammonium chloride,
36.15
57.31
62.00
29.91
38.40 46.50
57.21
75.84
97.14 102.90
122.37 130.00
100 mM Ammonium chloride,
80
60
25.02
40
15.27 19.79
1.30
20
58.33
124.47
75.95
0
0
20
40
60
80
Tim e (m in)
132.44
98.84 101.21
100
120
140
NL:
4.13E9
Base Peak
MS
yeast_120mi
ng_04
Yeast Protein Separation
RT: 0.00 - 147.01
140 mM Ammonium chloride,
52.66
32.77
100
NL:
4.05E9
Base Peak
MS
yeast_120mi
ng_05
80
18.84
60
46.16
44.10
57.14
40
12.57
20
1.57
58.03
Relative Abundance
0
100
75.86
87.56
96.28
121.87 129.57 137.22
NL:
3.24E9
Base Peak
MS
yeast_120mi
ng_06
41.06
180 mM Ammonium chloride,
80
26.85
22.22
60
40
13.34
32.88
20
50.93
54.17
12.54
2.55
0
100
17.01
57.88 75.96
128.39
87.63
89.93
124.39
132.41
NL:
1.02E9
Base Peak
MS
yeast_120mi
ng_07
18.52
220 mM Ammonium chloride,
80
60
27.73
1.34
40
1.89
20
42.07
30.91
50.18 57.70
14.82
75.86
76.99
70.04
87.67
97.96 113.09 129.14
134.87
0
0
20
40
60
80
Tim e (m in)
100
120
140
Yeast proteins
Reference
Score Hits
gi|129922|sp|P14828|PGK_KLULA PHOSPHOGLYCERATE KINASE gi|66895|pir||KIVKGL
65809.5 18 4 4 3 0
gi|6319594|ref|NP_009676.1| translational elongation factor EF-1 alpha; Te
56564.0 12 1 1 0 0
gi|119145|sp|P16017|EF1A_CANAL ELONGATION FACTOR 1-ALPHA (EF-1-ALPHA)
46500.4 0gi|1
11 1 1 0
gi|1172457|sp|P41757|PGK_CANMA PHOSPHOGLYCERATE KINASE gi|83923|pir||JT095
46303.5 12 1 0 0 0
gi|6324637|ref|NP_014706.1| Ribosomal protein L3 (rp1) (YL1); Rpl3p [Sacch 42592.4 10 0 1 1 0
gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae] gi|141268.3 10 2 1 0 0
gi|6319279|ref|NP_009362.1| Pyruvate kinase; Cdc19p [Saccharomyces cerevis38052.7 23 3 0 0 0
gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae] gi|136687.9 13 2 1 0 1
gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae] gi|134389.6 13 2 1 0 1
gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 34030.4 13 7 0 0 0
gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase; Pgk1p [Saccharomyc
26362.2 7 4 1 0 0
gi|6322790|ref|NP_012863.1| aldolase; Fba1p [Saccharomyces cerevisiae] gi| 26245.4 2 0 0 0 0
gi|6319279|ref|NP_009362.1| Pyruvate kinase; Cdc19p [Saccharomyces cerevis25546.9 9 0 0 0 0
gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 23954.0 7 1 0 0 1
gi|6325112|ref|NP_015180.1| sequence similar to Hes1p; Kes1p [Saccharomyce
23937.7 3 0 2 2 0
gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase; Pgk1p [Saccharomyc
23524.5 3 2 1 0 1
gi|6321609|ref|NP_011686.1| phosphatidylserine decarboxylase located in va 23254.1 2 0 1 3 4
gi|6322526|ref|NP_012600.1| phosphatidylinositol kinase homolog; Tor1p [Sa 21082.5 0 3 2 1 2
gi|14318479|ref|NP_116614.1| Actin; Act1p [Saccharomyces cerevisiae] gi|1121011.4 2 0 0 0 0
gi|12230852|sp|O74343|YH2X_SCHPO HYPOTHETICAL 76.4 KDA PROTEIN C1A4.09
20063.6 0IN1 1 1 1
gi|119336|sp|P00924|ENO1_YEAST ENOLASE 1 (2-PHOSPHOGLYCERATE 19580.1
DEHYDRATASE)
40000
gi|6324313|ref|NP_014383.1| Pbi2p [Saccharomyces cerevisiae] gi|124818|sp|19318.9 5 0 0 0 0
gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 19151.9 7 0 0 0 1
gi|6325331|ref|NP_015399.1| Transketolase 1; Tkl1p [Saccharomyces cerevisi 18932.7 5 1 0 0 0
gi|6321631|ref|NP_011708.1| Glyceraldehyde-3-phosphate dehydrogenase 3; Td18650.3 3 0 0 0 0
gi|6321977|ref|NP_012053.1| 6-phosphogluconate dehydrogenase; probable GND
18647.0 4 0 0 1 0
gi|7492246|pir||T40586 nucleolar protein involved in pre-rRNA processing 18417.0 6 8 12 10 8
gi|7491534|pir||T40003 hypothetical protein SPBC25H2.08c - fission yeast
18270.9 0 1 2 0 0
gi|6325016|ref|NP_015084.1| 82 kDa heat shock protein; homolog of mammalia18036.9 6 2 0 0 0
Entries
91 95 96 101 255 260 686 689 824 873 888 902 913 925 926 94
76 215 239 336 1020 1024 1159 1163 1338 1362 1518 1526 : 1
: 76 215 239 1020 1024 1159 1163 1338 1362 1518 1526 : 113
255 260 824 860 873 888 913 925 926 948 960 1050 : 994 : : :
133 138 143 383 387 468 471 482 484 1531 : : 386 : 1305 :
38 56 58 69 77 262 269 270 302 313 : 180 1413 : 163 : :
29 38 59 70 73 92 132 136 166 288 291 421 474 483 900 1121
8 11 39 42 129 377 396 486 488 590 802 1751 1814 : 1014 162
19 50 329 349 441 442 445 820 1028 1037 1434 1522 1548 : 34
49 113 116 212 215 336 567 608 804 1082 1275 1317 1320 : 12
185 187 307 313 404 1743 1785 : 109 373 1746 1804 : 1811 : :
266 269 : : : :
61 131 218 276 309 310 1005 1399 1404 : : : :
97 143 204 294 356 365 492 : 297 : : : 264
611 736 1234 : : 76 1362 : 215 1547 :
171 173 298 : 175 1060 : 1226 : : 378
475 1103 : : 404 : 397 451 1007 : 139 185 400 1607
: 7 80 770 : 1518 1690 : 1024 : 75 1020
372 384 : : : :
: 1603 : 847 : 1607 : 1590
177 260 302 313 : : : :
62 70 73 1878 1882 : : : :
208 211 213 215 1467 1609 1610 : : : : 1288
217 218 223 224 1328 : 420 : : :
1590 1603 1607 : : : :
647 1529 1530 1534 : : : 695 :
1075 1243 1276 1414 1508 1540 : 1009 1076 1247 1482 1585 1
: 143 : 133 138 : :
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
gi|6321007|ref|NP_011086.1| Transcriptional regulator which functions in m
4747.8 0 0 0 1 0
gi|6323292|ref|NP_013364.1| Ylr262c-ap [Saccharomyces cerevisiae] gi|21318 4746.0 0 1 0 0 1
gi|6322314|ref|NP_012388.1| Yjl147cp [Saccharomyces cerevisiae] gi|1353019 4744.7 0 0 0 1 0
gi|6324034|ref|NP_014104.1| Ynl295wp [Saccharomyces cerevisiae] gi|13531064744.4 0 1 0 0 0
gi|6324151|ref|NP_014221.1| Ribosomal protein S3 (rp13) (YS3); Rps3p [Sacc 4743.3 2 0 0 0 0
gi|6323691|ref|NP_013762.1|
4741.6 0 0 3 0 3
gi|6322806|ref|NP_012879.1| p58 polypeptide of DNA primase; Pri2p [Sacchar 4738.8 0 0 1 0 1
gi|1171671|sp|Q09711|NCS1_SCHPO HYPOTHETICAL CALCIUM-BINDING PROTEIN
4737.8 C18B1
00010
gi|7496455|pir||T19442 hypothetical protein C25A1.7a - Caenorhabditis eleg
4737.0 0 0 1 2 1
gi|83218|pir||S19440 hypothetical protein YCR029c - yeast (Saccharomyces c 4736.0 0 0 1 0 0
gi|6323487|ref|NP_013559.1| Ylr454wp [Saccharomyces cerevisiae] gi|13637324735.9 1 1 2 2 0
gi|6319642|ref|NP_009724.1|
4735.1 0 0 0 0 1
gi|6319850|ref|NP_009931.1| non-mitochondrial citrate synthase; Cit2p [Sac
4729.1 1 1 0 0 1
gi|6324552|ref|NP_014621.1| 3'-5' exoribonuclease complex subunit; Dis3p [
4728.2 0 0 1 1 0
gi|7490292|pir||T38695 conserved hypothetical protein SPAC3C7.09 - fission 4726.5 0 0 1 1 0
gi|6320126|ref|NP_010206.1|
4724.8 0 0 0 2 3
gi|1077218|pir||S49776 hypothetical protein YDR179w-a - yeast (Saccharomyc 4723.5 0 0 1 0 0
gi|1352297|sp|P48996|DP27_CAEEL CHROMOSOME CONDENSATION PROTEIN
4723.3
DPY-27
1 0 0 1gi|2
gi|6323875|ref|NP_013946.1| involved in silencing; Esc1p [Saccharomyces ce 4722.7 1 0 1 0 1
gi|1175464|sp|Q09796|YAA2_SCHPO HYPOTHETICAL 111.5 KD PROTEIN C22G7.02
4719.0 1IN0 1 0 1
gi|6323762|ref|NP_013833.1| Ymr115wp [Saccharomyces cerevisiae] gi|2497155
4717.5 0 0 0 1 0
gi|6322171|ref|NP_012246.1| Ribosomal protein L2B (L5B) (rp8) (YL6); Rpl2b 4715.9 1 1 0 0 0
gi|2495228|sp|Q12578|HIS7_CANGA IMIDAZOLEGLYCEROL-PHOSPHATE DEHYDRATASE
4715.9 0 1 0 0 (I0
gi|15213983|sp|O94489|EF3_SCHPO ELONGATION FACTOR 3 (EF-3) gi|7492556|pir|
4715.5 1 0 1 0 0
gi|6321594|ref|NP_011671.1| Cystathionine beta-synthase; Cys4p [Saccharomy4712.9 2 0 0 0 0
gi|6323722|ref|NP_013793.1| (putative) involved in sister chromosome cohes
4712.4 1 0 0 0 1
gi|113719|sp|P12807|AMO_PICAN PEROXISOMAL COPPER AMINE OXIDASE4712.2
(METHYLAMIN
46435
gi|6320232|ref|NP_010312.1|
4707.7 0 0 0 0 1
gi|6322088|ref|NP_012163.1|
4707.5 0 0 0 2 0
1624 1627 : 1625 : 652 : :
: : : 33 :
: 876 : : : 732
: : : 460 :
: 396 : : :
279 332 : : : :
: : 1877 1949 2145 : : 981 1314 1853
: : 971 : : 558
: : : 397 :
: : 1597 : 185 1645 : 1521
: : 50 : :
724 : 6 : 638 1305 : 359 1200 :
: : : : 224
646 : 1020 : : : 1619
: : 1637 : 329 :
: : 766 : 1441 :
: : : 342 712 : 598 637 1671
: : 50 : :
1196 : : : 482 : 122 896
1103 : : 1722 : : 792
1954 : : 294 : : 214
: : : 243 :
42 : 25 : : :
: 610 : : :
24 : : 326 : :
284 652 : : : :
561 : : : : 198
1168 1335 1362 1468 : 1169 1228 1471 1496 1499 1613
: : : : 202
: : : 116 1740 :
Protein # 1708
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
spectrometer
1
3
Pump 1
Sample valve
injector
6
RP 1
2
Column 1
SCX
IMAC
Injector
Sample loop
LCQ Deca XP
Plus- mass
spectrometer
4
5
Sample Pump
To Waste
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 flowthrough 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
RP2 column
Non-phosphorylated peptides
flow through IMAC column and
captured by and eluted from RP2
NL: 1.34E9
position for m/z=1031.7 on C2 column
RP1 column
Phosphorylated peptide (m/z=1031.7,
FQ*SEEQQQTEDELQDK)
captured by IMAC column, bound to
RP1, and eluted.
NL: 1.80E8
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
1+
2+
(M+2H)-49
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:
4.02E9
Base Peak
MS
A431_04040
2_01
36.47
11.84
20.80
974.2
8.17 652.2
1154.5
4
513.5
27.76
14.06
866.2
34.75
6.03
981.2
814.4
652.9
37.28 45.66
974.1 955.3 51.00 53.93 59.86 66.46 74.00 77.03
614.2 804.3 1179.0 971.0 1010.4 1024.2
NL:
20.34
34.70 37.53
7.25E8
896.2
970.7 974.2
31.70
Base Peak
MS
38.01
26.50 925.6
50.68
13.92
a431_04040
1335.0
784.4
496.2
2_02
10.95 581.5
566.9
42.02
52.10 54.90
1.21
65.23
73.06 78.78 85.51
322.0
522.3 716.3
712.6
369.3
494.5 371.0 371.0
NH Cl 0 mM
100
50
0
50
0
23.83
1154.7
100
50
1.36
712.6
20.32
13.92 896.1
464.4
0
22.25
820.7
50.28
991.6
31.16 37.57
1113.3 974.6
51.75
522.2
46.23
955.2
54.11
760.2
14.08
1.06
712.6 12.34 655.0
729.2
28.85
1079.1 36.47
814.4
45.61
955.3
10
20
30
40
50
Time (min)
60 mM
100
50
0
80 mM
100
50
0
100
1.35
50 712.5
85.00
370.9
NL:
1.26E9
Base Peak
MS
a431_04040
2_04
52.37
1043.7
0
0
68.69 74.10
1418.3 1010.4
40 mM
23.73
839.4
50
NL:
6.96E8
Base Peak
MS
a431_04040
2_03
20 mM
18.07
652.3
100
Relative Abundance
10 mM
100
NL:
35.16
50.21
9.00E8
947.6
991.8
18.73
30.76
Base Peak
893.3 19.50 832.0
MS
14.64
a431_04040
654.6
653.5
2_05
51.82
27.91
8.92
522.2
653.8
578.0
35.81 41.74
53.03 59.38 64.81 72.10 80.53 83.25
1286.3 322.0
522.6 842.3 371.0 390.8 370.9 370.9
NL:
23.93
1.10E9
797.2
31.93
Base Peak
MS
866.3
a431_04040
26.10
16.37
2_06
878.6
50.62
12.80 600.1
1.22
496.2
38.72 42.18
495.8
712.5
56.97 65.29 69.94 73.18 78.99 85.04
1103.9 322.0
315.9 369.3 508.7 508.6 370.9 370.9
60
100
0.82
712.4
80
17.43
15.47
480.8
478.7
50.63 52.17
32.76
496.3 522.2
932.6 38.09 42.17
56.86 72.03 73.04 80.31 86.81
894.7 321.9
315.9 508.5 508.7 370.9 370.9
0
0
10
20
160 mM
30.16
822.8
23.90
668.8
30
40
50
Time (min)
NL:
3.95E8
Base Peak
MS
a431_04040
2_07
52.55 59.49 69.19 72.16 80.72 83.45
522.4 842.4 1269.7 390.9 371.0 370.9
22.56
682.7
72.50 81.03 84.35
390.9 370.9 370.9
70
120 mM
51.72
50.30 522.3
496.2
36.50
38.62
900.1
894.5
9.30
630.8
0
50
56.98 62.62
315.9 563.3
24.01 27.32
697.0 889.5
11.49
417.3
33.02
932.8
60
70
80
NL:
1.04E9
Base Peak
MS
a431_04040
2_08
Automated 2D-LC-LC/MS-MS Analysis of Human
A431 Cell Proteins (continued)
RT: 0.00 - 87.02
15.05
506.3
100
1.20
712.5
50
13.02
9.31 503.1
452.8
Relative Abundance
0
15.60
473.0
41.68
322.1
31.88
29.42 569.4
677.9
1.14
712.6
100
1.91
712.5
50
50.26
496.3
200mM
59.03 62.99
886.5 347.9
72.08
391.0
82.29
370.8
NL:
2.46E8
Base Peak
MS
a431_04040
2_10
52.16
50.64 522.2
496.2
12.85
603.4
32.45
569.6
19.02
857.9
30.87
832.1
42.15
321.9
300mM
49.37
496.5
32.95
932.5
0
77.53 79.64 85.51
370.9 370.9 370.9
63.71
347.9
53.47
508.3
NL:
2.95E8
Base Peak
MS
a431_04040
2_11
50.36
991.7
1.09
712.4
100
NL:
4.11E8
Base Peak
MS
A431_04040
2_09
51.86
522.4
51.73
1043.5
2.00
712.5 9.27 15.78
446.4 824.2
50
0
25.96
769.1
32.17
569.7
500mM
41.92
321.9
59.23 63.24
886.4 348.0
81.51
72.13 78.98
370.9
390.9 370.9
NL:
2.70E8
Base Peak
MS
a431_04040
2_12
1.04
712.5
100
1.69
712.4
50
5.69
712.5
32.37
569.6
19.62
712.5
29.70
347.9
37.95
331.9
42.27
321.9
50.64
496.3
46.88
399.1
52.17
522.3
900mM
63.72
348.0
54.66
371.1
70.87
508.7 72.54
390.9
83.46
370.9
0
0
10
20
30
40
50
Time (min)
60
70
80
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 EGFStimulated 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 EGFtreated 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.
3.
ProteomeX (2D)
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
4.
5.
Collect the ICAT-peptide fractions and run LC-MS/MS.
ProteomeX (1D)
Data analysis by Bioworks 3.1
a) TurboSEQUEST for protein identification
b) XPRESS for relative quantitation
ProteomeX(2D)
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 cysteinecontaining 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.
Zoom In MS spectra
RT: 35.09 - 35.37
NL:
9.68E5
35.29
100
(+2) Charge D8
M/Z = 799.1 Signal = 0.986
m/z=
90
MS
35.25
80
Relative Abundance
70
Base Peak
35.33
35.20
798.6-799.6
60
50
35.16
40
30
20
35.16
10
0
100
90
(+2) Charge D0
80
50
70
40
60
30
M/Z = 794.9 Signal =1.09
NL:
1.09E6
Base Peak
35.33
D0/D8 = 1.1
35.20
794.4-795.4
MS
Using the highest MS intensity
m/z=
20
10
35.29
0
35.10
35.15
35.20
35.25
Time (min)
35.30
35.35
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
18
( O)
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
procedure
18O
labeling
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 modereject 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
% Relative Abundance
100
Serine
Phosphorylated
90
80
70
Serine Not
Phosphorylated
931.8 (MH2 - H3PO4)2+
60
50
Y’’12+1
40
30
Y’’10+1
20
10
922.7
764.6
452.5551.1
366.4
665.3
1311.5
1099.5
1410.6
1083.1
1591.51689.7
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
NL:
2.79E10
TIC MS
46.78
100
Glycopeptide region
90
80
31.08
MS from
Relative Abundance
70
60
44.66
1
2
5.32
50
40
Other region perform only MS and MS/MS
34.55
26.55 29.80
3
4
6.06
30
8.59
20
56.23
21.55
13.23
97.61
49.19
57.42
61.02
21.91
96.92
95.93
64.91
10
98.25
88.24
70.50 75.92 86.75
110.98
121.72
113.39
117.35
122.46
138.88
0
0
10
20
30
40
#583
50
RT: 15.57
60
70
Time (min)
80
90
100
110
120
130
AV: 1 NL: 7.23E8
T: + c Full ms [ 200.00-2000.00]
527.5
Glycopeptide region
Relative Abundance
MS scan
As
n
glycopeptide ion
15
217.0
Fu
N
20
(select to do MS/MS)
575.8
10
+3
5
234.1
286.8
1064.0
445.0
634.8
762.3
807.9
1060.7
1095.7
1267.6
1514.8
+2
1595.2
1780.2
0
200
400
600
800
1000
1200
m/z
1400
1600
1800
1987.9
2000
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
Asn
95
90
85
(select to do MS to 3)
80
75
70
65
Relative Abundance
60
55
50
+2
45
Fu
40
35
1185.8
30
25
20
+1
+1
15
10
5
+2
Fu
N
366.0
657.0
739.5
453.8
923.0
+2
Asn N
Fu
Asn
1413.1
Fu
1449.6
Fu
+2
Asn
Asn
1369.4
Asn
1003.5
966.8
1450.3
1478.9
1085.1
1845.7
1933.3
0
400
600
800
1000
1200
m/z
1400
1600
1800
2000
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
As
n
95
90
85
80
Fu
Fu
70
As
666.6
n
65
Relative Abundance
oxidized
+2
75
60
Asn
1479.2
Fu
As
n
50
+2
35
25
Asn
1537.6
1334.6
1185.7
Asn
740.3
Fu
Fu Asn
1085.2
Fu
30
Asn
1478.3
993.5
40
Fu
1011.3
55
45
(select to do MS to 4)
892.4
1987.5
528.0
20
551.2
15
Fu
768.6
586.1
10
1697.2
Asn
1460.4
930.3
1315.8
1859.1
5
0
400
600
800
1000
1200
m/z
1400
1600
1800
2000
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
Dehydro-alanine form
Further CNH2 loss on N-terminal
B6
95
(select to do MS to 5)
90
85
80
75
65
B7
C-T-S-Q-H-L-L-N-R
Peptide only
C-T-S-Q-H-L-L-N-R
70
Relative Abundance
B6
B5
SCH2COOH
1130.7
y
2
Y7
60
55
50
506.2
45
40
Y7
35
B7
30
25
20
B5 636.2
Y2492.2
/ Y6(+2)618.3
833.0
-H2O
Dehydro-alanine form
C-T-S-Q-H-L-L-N-R
-H2O
1053.4
823.4
798.0
1240.5
15
10
5
0
400
600
800
1000
1200
m/z
1400
1600
1800
2000
Summary of one glycopeptide fragmentation
pathway (a biantennary glycopeptide)
Fu
Fu
Fu
N
MS to 3
MS/MS
MS
Asn
Asn
Asn
(1267 +2)
Fu
211
210
(1064.4 +3)
(1450.6 +2)
Asn
(1186 +2)
Fu
Asn
LCQ-deca (nanospray)
(1085 +2)
Fu
Asn
(1105 +2)
Fu
LCQdecaXP (microspray)
MS to 4 or 5
CTSQHLLNR(1333 +1)
Peptide only (1131 +1)
Asn
(1004 +2)
Asn
(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
496.1
100
Base Peak
80
60
Full Scan MS
40
20
0
16
18
20
22
Time (min)
730.4
*
100
24
80
990.6
400
600
800
m/z
Full Scan MS2
389.2
261.1 *
40
*
616.4
233.1
*
502.4 *
20
732.4
0
200
400 600
800
m/z
Full Scan MS3
@233.1
1200
713.4
60
120.2
1000
Full Scan MS3
@730.4
1000
86.1
389.2
714.5
502.3
121.2
400
100
200
300
m/z
400
233.1
Full Scan MS3
@261.1
599.3
354.1
261.2
226.1
Full
Scan MS3
@389.2
0 100
200
300
m/z
500
Full Scan MS3
@616.4
581.2
129.1
400
1200
372.2
243.0
234.1
800
m/z
200
400
m/z
600
200 400 600 800 1000
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!