Quantitative Proteomics - Ohio State University · Workflow of TMT™ Reagents (up to 10 Samples)...
Transcript of Quantitative Proteomics - Ohio State University · Workflow of TMT™ Reagents (up to 10 Samples)...
Quantitative Proteomics
Liwen ZhangMass Spectrometry and Proteomics Facility
The Ohio State University
Summer Workshop 2018
Quantitative ProteomicsQuantitation in proteomics has become a popular area in recent proteomics research with the development of quantitation techniques such as DIGE, SILAC, ICAT, iTRAQ and Label Free.
•Difference Gel Electrophoresis•Gel based using cy-dye chemistry.
•Isobaric tag for relative and absolute quantitation – iTRAQ/TMT• is a non-gel based technique used to identify and quantify proteins/peptides from different sources in one single experiment by using isotope coded covalent tags that will label the N-terminus and side chain amines of peptides from protein digestions.
•Stable isotope labeling by amino acids in cell culture – SILAC• is a non-gel based approach for in vivo incorporation of a label into proteins for MS quantitative proteomics. It relies on metabolic incorporation of a given 'light' or 'heavy' form of the amino acid into the proteins.
•Label Free Quantitation•It has been observed the chromatographic peak areas and number of spectra/peptides observed for a protein in a LC/MS/MS run is correlated with the concentration of that particular protein.
DIGE (Difference Gel Electrophoresis)
Spot Volume = [spot 1 on treated]/[spot 1 on standard]
DIGE (Difference Gel Electrophoresis)
In Gel Digestion, LC/MSMS, MASCOT
DIGE (Difference Gel Electrophoresis)
high sensitivity
linearity of the dyes utilized
straightforward significant reduction of experiment error
High reproducibility
Pros and Cons to DIGE
300 – 500 µg of total protein is required for each biological replicate
Requires high resolution 2D gels
Not ideal for membrane proteins
Not ideal for serum type samples
Some protein spots identify more than one protein or do not have enough protein to identify the spot
Labor Intensive
iTRAQTM (Applied Biosystems)
https://www.researchgate.net/figure/283567390_fig1_Fig-1-Chemical-structures-for-iTRAQ-a-4-plex-and-b-8-plex-isobaric-tags
Chemistry of iTRAQ™ Reagents
Overview of iTRAQ™Reagents Methodology
Labeling Lys and N-terminus
Up to 8 samples
Fractionation offers more identification
Trypsin digestion
Cell Culture + Tissue
Isobaric tag for relative and absolute quantitation
Group IS Normal Mod Mild Mix1 IS 1 (126) 2004 (127) 2041 (128) 2043 (129) 2069 (130)2 IS 2 (126) 2007 (131) 2042 (127) 2040 (128) 2066 (129)3 IS 3 (126) 2008 (130) 2065 (131) 2070 (127) 2037 (128)4 IS 4 (126) 2063 (129) 2046 (130) 2038 (131) 2014 (127)5 IS 5 (126) 2015 (128) 2068 (129) 2071 (130) 2036 (131)6 IS 6 (126) 2016 (127) 2067 (128) 2062 (130) 2035 (131)
Dry Eye Studies: 4 Groups: Normal, Moderate Dry Eye, Mild Dry Eye, Mixed Dry Eyes
100 300 500 700 900 1100 1300 1500m/z 19001700
232.12y2
331.31y3
559.25y5
674.35y6
821.55y7
968.30y8
1131.44y9
1245.38y10
1344.23y11
1401.32y121458.29
y131605.40
y141706.27
y151834.52
y16
487.24b2
588.18b3
792.12b5 849.14
b6948.30
b71062.54
b81225.15
b91372.42
b101519.43
b111634.48
b121733.70
b131862.61
b14735.20
b4359.16
b1
Peptide Sequence:EQTFGGVNYFFDVEVGRProtein Name : Human Cystatin-S
Observed m/z= 1097.02702+
Theoretical m/z= 1097.04432+
124 126 128 130 132 134m/z05
101520253035404550556065707580859095
100
Rel
ativ
e A
yund
ance 126
127 129128
131Ratio 126:127:128:129:130:131=1:0.30:0.09:0.25:0.00:1.49
99.17Val
227.94 (129+99)Glu-Val
115.10Asp
147.20Phe
146.75Phe
163.14Tyr
113.94Asn
98.85Val
57.09Gly
56.97Gly
147.11Phe
100.87Thr
128.25Gln
99.22Val
115.05Asp
147.01Phe
147.27Phe
162.61Tyr
114.24Asn
99.16Val
56.92Gly
57.02Gly
147.02Phe
100.94Thr
128.08Gln
128.91Glu
iTraq MASCOT Results
Protein Name
DE/NDE (Ratio (# of Detection, p value))
FunctionMild/Normal Mod/Normal Mix/Normal
Aldehyde dehydrogenase, dimeric NADP-preferring OS=Homo sapiens (2.41, 6, 0.0030) (1.69, 6, 0.0471) oxidoreductase activity
Apolipoprotein A-I OS=Homo sapiens (1.75, 6, 0.0362) transporter activity; protein binding; enzyme regulator activity; lipid binding
Cystatin-S OS=Homo sapiens (0.51, 6, 0.0014) (0.43, 6, 0.0005) (0.60, 6, 0.0283) enzyme regulator activity
Deleted in malignant brain tumors 1 protein OS=Homo sapiens (0.60, 6, 0.0472) signal transducer activity; protein binding; bacterial cell surface binding
Ezrin OS=Homo sapiens (1.79, 6, 0.0101) protein binding; binding
Hemopexin OS=Homo sapiens (2.15, 6, 0.0384) (2.11, 6, 0.0486) binding; transporter activity; ion binding
Haptoglobin OS=Homo sapiens (1.97, 6, 0.0474) catalytic activity; protein binding; peptidase activity
Ig alpha-1 chain C region OS=Homo sapiens (0.59, 6, 0.0113) antigen binding; protein binding
Ig gamma-1 chain C region OS=Homo sapiens (1.94, 6, 0.0266) antigen binding; protein binding
Ig gamma-2 chain C region OS=Homo sapiens (1.87, 6, 0.0369) antigen binding
Ig gamma-3 chain C region OS=Homo sapiens (2.08, 6, 0.0408) antigen binding
Extracellular glycoprotein lacritin OS=Homo sapiens (0.28, 6, 0.0059) (0.23, 6, 0.0003) (0.48, 6, 0.0428) protein binding; extracellular matrix binding
Putative lipocalin 1-like protein 1 OS=Homo sapiens (0.43, 6, 0.0026) (0.39, 6, 0.0002) (0.50, 6, 0.0119) binding; transporter activity
Lipocalin-1 OS=Homo sapiens (0.33, 6, 0.0030) (0.32, 6, 0.0003) (0.43, 6, 0.0188) binding; transporter activity; protein binding; enzyme regulator activity
Lysozyme C OS=Homo sapiens (0.33, 6, 0.0019) (0.28, 6, 0.0031) catalytic activity; hydrolase activity; protein binding
Polymeric immunoglobulin receptor OS=Homo sapiens (0.51, 6, 0.0290) (0.43, 6, 0.0016) protein binding
Prolactin-inducible protein OS=Homo sapiens (0.36, 6, 0.0004) (0.38, 6, 0.0008) (0.58, 6, 0.0409) protein binding
Proline-rich protein 1 OS=Homo sapiens (0.44, 6, 0.0023) (0.39, 6, 0.0056) enzyme regulator activity
Proline-rich protein 4 OS=Homo sapiens (0.41, 6, 0.0042) (0.34, 6, 0.0081)
Secretoglobin family 1D member 1 OS=Homo sapiens (0.28, 6, 0.0032) (0.21, 6, 0.0006) (0.41, 6, 0.0169) binding
Mammaglobin-B OS=Homo sapiens (0.34, 6, 0.0076) (0.26, 6, 0.0009) (0.43, 6, 0.0215) steroid binding; binding; hormone binding
Serotransferrin OS=Homo sapiens (2.02, 6, 0.0429) protein binding; ion binding
Lactotransferrin OS=Homo sapiens (0.33, 6, 0.0019) (0.34, 6, 0.0072) peptidase activity; ion binding; pattern bindingcarbohydrate binding; hydrolase activity; protein binding
Vitamin D-binding protein OS=Homo sapiens (1.65, 6, 0.0449) (1.87, 6, 0.0206) transporter activity; steroid binding; vitamin binding;: protein binding
Zinc-alpha-2-glycoprotein OS=Homo sapiens (0.40, 6, 0.0047) (0.36, 6, 0.0029) (0.59, 6, 0.0502) lipid binding; carboxylic acid binding; hydrolase activity; transporter activity
Zymogen granule protein 16 homolog B OS=Homo sapiens (0.44, 6, 0.0383) carbohydrate binding
iTraq Results
Chemistry of TMT™ 6Plex Reagents
Figure Provided Courtesy of Thermo Fisher Scientifics
Figure Provided Courtesy of Thermo Fisher Scientifics
Workflow of TMT™ Reagents (up to 10 Samples)
TMT Proteome Discoverer Results
Description Coverage # Peptides# PSMs # Unique P Ratio:127/126 Ratio:128/126 Ratio:129/126 Ratio:130/126 Ratio:131/126Vimentin OS=Homo sa 92.27468 70 764 68 1.073 1.296 1.159 1.324 0.489Glyceraldehyde-3-pho 90.44776 33 195 33 1.463 1.884 1.73 1.632 0.548Actin, cytoplasmic 1 OS 90.4 33 355 18 1.322 1.489 1.411 1.411 0.541Thioredoxin OS=Homo 88.57143 8 31 8 1.431 1.636 1.647 2.045 0.55410 kDa heat shock prot 84.31373 11 19 11 1.118 1.143 0.98 0.891 0.395Ubiquitin carboxyl-ter 84.30493 11 16 11 1.689 1.715 2.015 1.71 0.546Tubulin beta-4B chain 82.92135 31 169 4 1.521 1.78 1.621 1.438 0.629Alpha-enolase OS=Hom 82.48848 36 161 32 1.505 1.903 1.747 1.596 0.509Galectin-1 OS=Homo sa 82.22222 13 46 13 1.123 1.391 1.291 1.562 0.571Tubulin beta chain OS= 82.20721 30 176 5 1.502 1.957 1.631 1.439 0.638Triosephosphate isom 79.37063 22 64 22 1.755 1.741 1.89 1.554 0.532Annexin A2 OS=Homo 79.05605 34 111 34 1.118 1.403 1.267 1.606 0.563ATP synthase subunit b 76.93762 25 56 25 1.183 1.222 1.258 1.494 0.491Tubulin alpha-1A chain 76.7184 28 162 3 1.663 2.091 1.901 1.948 0.682Tubulin alpha-1C chain 76.6147 28 131 4 1.499 1.978 1.723 2.055 0.78960S acidic ribosomal pr 76.52174 6 19 6 1.233 1.452 1.415 1.317 0.45Calmodulin OS=Homo 76.51007 7 83 7 1.336 1.246 1.288 1.581 0.49Myosin light polypepti 76.15894 9 35 7 1.362 1.437 1.381 1.434 0.547Glutathione S-transfer 75.71429 8 35 8 1.514 1.898 1.888 1.951 0.585Phosphoglycerate mut 75.59055 16 34 16 1.632 1.69 1.745 1.462 0.516Pyruvate kinase PKM O 75.32957 42 155 42 1.425 1.961 1.711 1.824 0.568SH3 domain-binding gl 74.19355 4 23 4 1.65 1.583 1.776 1.473 0.524Peptidyl-prolyl cis-tran 71.51515 12 36 12 1.379 1.657 1.466 1.147 0.417Profilin-1 OS=Homo sa 70.71429 11 38 11 1.389 1.975 1.723 1.954 0.595Myosin regulatory ligh 70.17544 11 31 8 1.395 1.571 1.543 2.07 0.685Fructose-bisphosphate 70.05495 25 57 25 1.57 1.613 1.609 1.369 0.53214-3-3 protein zeta/de 68.57143 19 51 17 1.442 1.806 1.747 2.048 0.62
TMT Proteome Discoverer Results
Pros and Cons to iTRAQ/TMT
Requires less total protein than DIGE (50 µg or less)
One experiment can determine fold change, protein ID and Post translational modification
Up to 8-10 groups can be compared at the same time
Samples can come from both tissue or cell culture
• Mass Spectrometry requirements are rigorousHigh resolutionAdvanced LC chromatographyLong instrument timePQD/HCD needed on IT instrument
• Produces enormous amount of data
• Requiring detailed bioinformatic analysis
• Expensive
SILAC (Stable isotope labeling by amino acids in cell culture)
Regular Media (Lys Light)
No Drug
Lys-depleted Media(Lys Heavy)
Drug Treatment
Combination/Extraction
Change =
Digestion/Peptide Identification
Fractionation/IP
Heavy Isotopic Labeled Amino Acids:
L-Lysine: 13C6, (+6Da), 13C6/15N2 (+8Da), 13C6/15N2/D9 (+17Da), 15N2/D9 (+11Da), D4 (+4Da)
L- Arginine: 13C6 (+6Da), 13C6/15N4 (+10Da), 13C6/15N4, D7 (+17Da),15N4/D7 (+11Da)
L-Tyrosine: 13C9 (+9Da)
1D or 2D LC/MSMS
Optimization of Heavy Amino Acids IncorporationS G R G K5 G G K8 G L G K12 G G A K16 R H R K20 V L R D N I Q G I T K31 P A I R R
L A R R G G VK44 R I S G L I Y E E T R G V L K59 V F L E N V I R D A V T Y
T E H A K77 R K79 T V T A M D V V Y A L K91 R Q G R T L Y G F G G
∆ M =4*11= 44
11353
11396
11309
11279
11351
11393
11200 11300 11400 11500 11600
Mass
17881_13Days #21693-22317 RT: 127.45-130.89 AV: 100 NL: 6.03E5T: FTMS + c ESI Full ms [350.00-2000.00]
920 940 960 980 1000 1020 1040 1060 1080 1100m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative A
bundance
935.19
944.68
1012.47
907.49
1082.79984.88
1044.02
945.93
982.84908.801086.07
1014.48
1047.03948.171001.85966.48 1057.00 1064.50924.47914.92
1032.53
1078.08
1041.70
957.65 979.78 1091.02
930.44 1009.11
1023.84
994.55
987.00
1095.56
K* =13C6/15N2 labeled LysR* =13C6/15N4 labeled Arg
Cell Culture Labeled for 4 days
Cell Culture Labeled for 7 days
SILAC Proteome Discoverer
17872_1_7 #1858-1959 RT: 22.95-23.41 AV: 13 NL: 5.19E4T: FTMS + p ESI Full ms [350.00-2000.00]
660 662 664 666 668 670 672 674 676 678m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative
Abunda
nce
663.38
672.39
663.88
672.90
664.39
673.40
675.40671.89
675.90664.89673.90667.18 670.41661.29
674.88
662.83 676.40 679.39668.18665.35 678.37666.42 668.89
18Da18Da
∆ M =10+8= 18Da
DNIQGITKPAIR DNIQGITK*PAIR*
K* =13C6/15N2 labeled LysR* =13C6/15N4 labeled Arg
17872_1_7 #1902 RT: 23.14 AV: 1 NL: 1.01E3T: ITMS + c ESI d Full ms2 [email protected] [170.00-1340.00]
200 300 400 500 600 700 800 900 1000 1100 1200m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relativ
e Abun
dance
685.43
606.03456.31
855.50
654.58
492.54 549.04
983.57
742.11511.08 798.47229.90 342.92 584.37
1151.56696.36298.04 428.40358.01 1168.47838.40784.19 1117.35921.24 1027.74279.94 423.46
873.56
615.20
703.42
663.81466.34
1001.27230.07558.07
602.24501.42 816.61342.89725.30
1143.22255.27
983.84825.94775.75 1012.89298.12 411.46
1068.81 1195.44975.96
MSMS of DNIQGITK*PAIR* MSMS of DNIQGITKPAIR
Ratio=Heavy/Light=1:1
Accession Description Ratio: Heavy/Light
Sample 1 Sample 2
P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens 0.096 0.049
P22314 Ubiquitin-like modifier-activating enzyme 1 OS=Homo sapiens 0.106 0.182
P23526 Adenosylhomocysteinase OS=Homo sapiens 0.250 0.151
P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 0.257 0.265
P52597 Heterogeneous nuclear ribonucleoprotein F OS=Homo sapiens 0.260 0.254
O43175 D-3-phosphoglycerate dehydrogenase OS=Homo sapiens 0.262 0.276
P31943 Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens 0.284 0.337
P23771 Trans-acting T-cell-specific transcription factor GATA-3 OS=Homo sapiens 0.295 0.379O00299 Chloride intracellular channel protein 1 OS=Homo sapiens 0.306 0.316
P07741 Adenine phosphoribosyltransferase OS=Homo sapiens 0.319 0.343
Q14103 Heterogeneous nuclear ribonucleoprotein D0 OS=Homo sapiens 0.359 0.390
Q9BQE3 Tubulin alpha-1C chain OS=Homo sapiens 0.380 0.404
P13639 Elongation factor 2 OS=Homo sapiens 0.391 0.609
P31949 Protein S100-A11 OS=Homo sapiens 0.394 0.626
P08107 Heat shock 70 kDa protein 1A/1B OS=Homo sapiens 0.412 0.435
P11586 C-1-tetrahydrofolate synthase, cytoplasmic OS=Homo sapiens 0.415 0.835
P23246 Splicing factor, proline- and glutamine-rich OS=Homo sapiens 0.418 0.387
P31942 Heterogeneous nuclear ribonucleoprotein H3 OS=Homo sapiens 0.424 0.382
P58546 Myotrophin OS=Homo sapiens 0.429 0.420
P11021 78 kDa glucose-regulated protein OS=Homo sapiens 0.442 0.514
P00558 Phosphoglycerate kinase 1 OS=Homo sapiens 0.450 0.574
P61978 Heterogeneous nuclear ribonucleoprotein K OS=Homo sapiens 0.472 0.445
P62826 GTP-binding nuclear protein Ran OS=Homo sapiens 0.475 0.496
P35080 Profilin-2 OS=Homo sapiens 0.484 0.409
O60506 Heterogeneous nuclear ribonucleoprotein Q 1.198 1.051
Q99623 Prohibitin-2 OS=Homo sapiens 1.222 0.706
Q06830 Peroxiredoxin-1 OS=Homo sapiens 1.251 1.183
P62244 40S ribosomal protein S15a OS=Homo sapiens 1.265 1.124
P07355 Annexin A2 OS=Homo sapiens 1.299 1.194
P28072 Proteasome subunit beta type-6 OS=Homo sapiens 1.302 1.133
P21333 Filamin-A OS=Homo sapiens 1.310 1.243
SILAC Proteome Discoverer
SILAC MaxQuant
Protein IDs H/L H/L normalized variability [ H/L normalized #1 H/L normalized #2 H/L normalized #3 H/L normalized #4 H/L variability #1 H/L variability #2 H/L variability #3 H/L variability #4sp|O75083|WDR1_HUMAN 0.73868 0.49787 16.506 0.51647 0.47227 0.49776 0.42986 22.459 16.166 23.143 15.822sp|P00352|AL1A1_HUMAN 0.56922 0.41693 10.611 0.45488 0.41005 0.42585 0.39198 8.264 9.4221 18.585 36.193sp|P01903|DRA_HUMAN 0.52018 0.38994 16.517 0.40177 0.45355 0.36485 0.38588 23.371 7.0303 16.759 13.14sp|P01911|2B1F_HUMAN;sp 0.67564 0.44803 19.404 0.47696 0.51286 0.37077 0.40544 20.528 8.4054 43.195 17.842sp|P06733|ENOA_HUMAN;s 0.60635 0.42854 16.788 0.47056 0.47441 0.40585 0.39676 15.9 29.008 16.777 16.347sp|P07437|TBB5_HUMAN;sp 0.61284 0.42665 20.77 0.42878 0.36437 0.33858 0.43109 17.86 62.69 53.347 35.269sp|P08865|RSSA_HUMAN 0.76633 0.49336 14.318 0.55719 0.50864 0.45037 0.47984 15.75 11.125 4.0767 9.9245sp|P10412|H14_HUMAN;sp| 0.24239 0.17842 19.985 0.20851 0.19008 0.17122 0.1608 12.001 20.446 18.897 37.116sp|P14550|AK1A1_HUMAN 0.4772 0.33042 19.756 0.33151 0.45207 0.36085 0.35049 21.881 45.956 28.25 10.978sp|P14618|KPYM_HUMAN;s 0.69122 0.49247 18.124 0.51133 0.52859 0.47487 0.46953 18.925 75.637 73.303 18.653sp|P15121|ALDR_HUMAN 0.61551 0.45403 11.468 0.52463 0.49678 0.4284 0.41575 20.116 17.17 12.841 11.27sp|P22626|ROA2_HUMAN 0.69888 0.48943 17.553 0.48363 0.65696 0.48906 0.58803 18.879 20.255 19.143 23.764sp|P24534|EF1B_HUMAN 0.5565 0.37586 11.979 0.38499 0.34737 0.30052 0.38896 15.25 18.88 17.484 10.005sp|P26447|S10A4_HUMAN 0.68422 0.48022 14.233 0.56271 0.54325 0.42162 0.5152 14.47 11.022 8.8936 13.44sp|P28068|DMB_HUMAN 0.6218 0.43181 11.582 0.46229 0.51408 0.41554 0.41403 17.433 7.709 6.5076 2.8015sp|P35754|GLRX1_HUMAN 0.52424 0.40059 12.129 0.49731 0.5024 0.31936 0.39223 20.812 16.575 15.126 16.034sp|P50552|VASP_HUMAN 0.1523 0.11366 10.3 0.12016 0.096602 0.11853 0.097167 18.344 13.171 8.6084 15.248sp|P52566|GDIR2_HUMAN 0.52698 0.37307 9.9274 0.37773 0.41031 0.35531 0.34494 12.827 15.699 10.656 17.545sp|P53634|CATC_HUMAN 0.63824 0.44043 18.262 0.52397 0.54729 0.35123 0.40655 23.993 28.347 31.433 12.961sp|P59998|ARPC4_HUMAN 0.64433 0.44187 7.2632 0.47422 0.41861 0.42377 0.41194 9.033 11.62 2.6723 5.9446sp|P60866|RS20_HUMAN 0.659 0.47304 13.95 0.59757 0.5963 0.43103 0.44341 9.0905 9.4678 23.935 14.586sp|P60953|CDC42_HUMAN 0.62253 0.42021 16.084 0.42021 0.36152 0.32089 0.35312 15.44 6.6748 12.261 21.515sp|P62826|RAN_HUMAN 0.33754 0.23408 9.6979 0.25069 0.28194 0.20331 0.25885 52.033 35.87 10.805 40.448sp|P67809|YBOX1_HUMAN;s 0.60934 0.46068 15.177 0.52521 0.50314 0.40425 0.46776 24.83 1.7427 16.798 2.7154sp|P68363|TBA1B_HUMAN;s 0.73028 0.48465 8.7514 0.51455 0.49593 0.45062 0.4635 7.8559 14.224 7.6826 5.6296sp|P84077|ARF1_HUMAN;sp 0.56325 0.41636 6.6611 0.48001 0.43235 0.40981 0.395 11.442 8.5717 0.7576 10.307sp|Q14019|COTL1_HUMAN 0.58209 0.38527 8.3687 0.41231 0.43285 0.35975 0.38295 6.9712 18.566 15.06 8.6241sp|Q16555|DPYL2_HUMAN 0.6755 0.4699 17.226 0.56152 0.48393 0.4345 0.43068 22.794 19.963 21.804 2.377sp|Q9UL46|PSME2_HUMAN 0.68009 0.46958 9.9733 0.54606 0.50083 0.45211 0.45216 2.9977 11.18 2.9997 7.4181
SILAC: Pros and Cons
Expensive
Special data analysis platform
Samples come from cell cultures
Time consuming
One experiment can determine fold change, protein ID and Post translational modification
Up to several groups can be compared at the same time
Mixing at the beginning, allow more sample preparation steps
significant reduction of experiment error
High reproducibility
doesn’t use a stable isotope containing compound tochemically label the protein. It can determine the relativeamount of proteins in two or more biological samples bycomparing peptide peak areas or spectral counting.
Label-free quantification
# b Seq. y #1 58.0287 G 132 171.1128 L 1362.7515 123 284.1969 L 1249.6674 114 413.2395 E 1136.5834 105 528.2664 D 1007.5408 96 641.3505 L 892.5138 87 698.3719 G 779.4298 78 861.4353 Y 722.4083 69 976.4622 D 559.3450 5
10 1075.5306 V 444.3180 411 1174.5990 V 345.2496 312 1273.6674 V 246.1812 213 K 147.1128
GLLEDLGYDVVVK
CASP4_MOUSECaspase-4OS=Mus musculus
LC/MSMS
Data Dependent Top5 Method
Data Dependent Acquisition and Dynamic Exclusion 30143_WT_LEG #14457-14668 RT: 88.19-89.24 AV: 19 NL: 3.22E5T: FTMS + c ESI Full ms [350.00-2000.00]
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative
Abunda
nce
743.89
649.66
997.60
736.42
499.30 729.64
584.62
758.15
562.36 643.41 788.92
445.12958.96
601.571063.19
947.46417.92 1044.181163.58538.31 668.66 876.43683.33
991.52
837.38480.61639.64
912.40817.43
1007.50
1193.31
508.31
1077.51 1154.23
724.35
1
32
54
67
8
MSMS
30143_WT_LEG #14664 RT: 89.25 AV: 1 NL: 1.39E3T: ITMS + c ESI d Full ms2 [email protected] [190.00-2000.00]
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z
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Rel
ativ
e A
bund
ance
898.13712.51
991.16
962.47776.52
918.39926.34
878.32589.17
638.90560.70
869.18734.69
525.26 693.22468.12 766.40 816.38
850.10660.95 1019.05
1048.37617.69
1167.301120.23424.27454.82
1092.28
30143_WT_LEG #14306 RT: 87.35 AV: 1 NL: 7.47E2T: ITMS + c ESI d Full ms2 [email protected] [260.00-2000.00]
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z
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Rel
ativ
e A
bund
ance
907.18
945.07624.99
738.98 850.52950.34
497.15722.80 767.95 1037.29898.17693.97 1167.831066.54
963.19833.56468.92 1136.33668.46601.35 1196.351022.35 1101.94418.44 511.27 579.50
30143_WT_LEG #14360 RT: 87.64 AV: 1 NL: 1.09E3T: ITMS + c ESI d Full ms2 [email protected] [140.00-1660.00]
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z
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Rel
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ance
622.91
679.63
515.34
819.15
557.39
510.16
715.31651.39566.39 1001.25
466.65574.64
932.45
540.92409.65 724.31501.05420.13 611.22 809.47 1019.31 1132.30737.84 1100.22793.33 873.75
30143_WT_LEG #14467 RT: 88.20 AV: 1 NL: 4.03E3T: ITMS + c ESI d Full ms2 [email protected] [190.00-1485.00]
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z
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1002.30
648.24
761.20 824.14
644.32
874.36711.34
1158.45
1097.28727.35 984.16470.06
442.20 598.03487.98
693.59805.93580.22509.03 622.06
562.69 919.63795.91 1193.021080.75868.80 967.13 1118.91424.11 1068.94
30143_WT_LEG #14359 RT: 87.63 AV: 1 NL: 3.35E2T: ITMS + c ESI d Full ms2 [email protected] [215.00-2000.00]
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Rel
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546.11
762.93 817.42
417.30 617.37 696.97878.39
706.20746.46
660.79
711.89822.64
842.66
489.08 869.10
641.63 807.75 854.16674.05 725.63
799.31881.19566.84 599.46540.10
580.62 886.21526.61
432.28
12 3
45
MS Cycle: 0.06SLC Peak Width >12S
Dynamic Exclusion
Make MSMS for the lower abundant ions possible!!
Digestion LC/MS
MS/MS
Proteomics Work Flow
Peak Area/Ion IntensityMASCOT
ProteomeDiscovererMaxQuant
Spectral CountingMASCOT/Scaffold
ProteomeDiscovererMaxQuant
Relative Concentration (emPAI)MASCOT
Label-free quantification by Spectra CountingCondition A Condition B
Trypsin Digestion LC/MSMS Analysis
Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics. 2011 Feb;11(4):535-53
Label Free Quantitation by Spectral CountingRelative protein quantification is achieved by comparing the number ofidentified MS/MS spectra from the same protein in each of multipleLC/MS/MS
Hongbin Liu, Rovshan G. Sadygov and John R. Yates, III, A Model for Random Sampling and Estimation of Relative Protein Abundance in Shotgun Proteomics Anal. Chem. 2004, 76, 4193-4201
# of reports = # of Treatment Conditions X # of Bio-replicates
Scaffold
Label Free Quantitation by Spectral Counting
Spectral Counting Results-Scaffold
Conditions
Bio-Replicates
P-Value
# of Spectra
Spectral Counting Results-Scaffold
Spectral Counting Results-Scaffold
Spectral Counting Results
Fundamentally, MS1-based measurements are more accurateand precise than spectral counting with a better linear dynamicrange. This arises due to a number of weaknesses of spectralcounting:
•No direct measurement of peptide ion properties•The response in terms of spectra per peptide ion is not constant acrossdifferent features.•The linear dynamic range of the method can be limited by saturation effects.•Dynamic exclusion methods, designed to improve DDA coverage, can also affectthe response.•There is a stochastic aspect to DDA sampling, hampering reproducibility; DDAsampling is also biased towards more abundant species, for this reason.
Label Free Quantitation by Precursor Ion Peak Area/Intensity
Label Free Quantitation--MaxQuant
Protein IDs #1-1 #1-2 #1-3 #1-4 #2-1 #2-2 #2-3 #2-4 Inten. 1_1 Inten. 1_2 Inten. 1_3 Inten. 1_4 Inten. 2_1 Inten. 2_2 Inten. 2_3 Inten. 2_4 Ratio(Intensity) Ratio (# of Spectralsp|Q8WZ42|TITIN_HUMAN 1404 1355 1183 1407 1323 1169 1272 1285 4.768E+09 4.452E+09 4.406E+09 4289400000 4.65E+09 2.877E+09 4.864E+09 4.163E+09 0.92 0.94sp|P13533|MYH6_HUMAN 202 193 189 199 193 173 201 180 1.494E+10 1.652E+10 1.107E+10 1.2969E+10 1.28E+10 7.563E+09 1.279E+10 1.106E+10 0.80 0.95sp|Q09666|AHNK_HUMAN 145 162 135 162 156 165 144 157 244500000 291410000 366210000 228120000 3.46E+08 500100000 288320000 319630000 1.29 1.03sp|P11532|DMD_HUMAN 129 116 102 120 112 91 106 113 160600000 152530000 170760000 137080000 1.74E+08 113320000 156400000 146500000 0.95 0.90sp|P98160|PGBM_HUMAN 112 123 111 119 117 130 124 117 339010000 334290000 332860000 333350000 4.99E+08 686210000 652970000 456730000 1.71 1.05sp|Q15149|PLEC_HUMAN;sp| 107 104 74 110 92 105 94 105 74806000 81093000 91831000 90837000 98467000 146260000 91691000 126970000 1.37 1.00sp|Q13813|SPTN1_HUMAN;RE 107 99 87 114 110 112 99 104 150860000 169550000 220710000 168010000 1.98E+08 236350000 186650000 157150000 1.10 1.04sp|Q14204|DYHC1_HUMAN 83 90 58 98 83 91 69 92 60618000 50595000 71894000 61528000 77399000 77761000 65451000 73120000 1.20 1.02sp|P12111|CO6A3_HUMAN 89 90 86 104 104 106 96 106 418240000 514340000 732980000 916810000 9.94E+08 1.508E+09 744710000 1.361E+09 1.78 1.12sp|Q14315|FLNC_HUMAN 92 93 71 99 86 78 92 95 255290000 252640000 200680000 354940000 2.43E+08 186680000 342080000 438940000 1.14 0.99sp|Q92736|RYR2_HUMAN;sp| 92 67 35 74 72 39 59 64 72914000 47014000 52146000 49132000 71266000 28427000 48761000 47980000 0.89 0.87sp|P35555|FBN1_HUMAN;sp| 86 75 89 95 94 98 82 88 742290000 373410000 693080000 923520000 2.09E+09 1.083E+09 672790000 930820000 1.75 1.05sp|Q01082|SPTB2_HUMAN;sp 72 75 64 78 78 87 73 77 96860000 108340000 131470000 119770000 1.4E+08 196680000 144500000 120480000 1.32 1.09sp|P35579|MYH9_HUMAN 72 60 54 70 59 67 54 56 97628000 82407000 90592000 93607000 1.48E+08 176340000 95382000 111960000 1.46 0.92sp|P52179|MYOM1_HUMAN 82 74 73 82 76 67 73 70 274290000 252840000 217130000 216800000 2.3E+08 121700000 216560000 178280000 0.78 0.92sp|P15924|DESP_HUMAN 72 66 62 69 63 49 60 66 85822000 79337000 126050000 83554000 98907000 54058000 115090000 101580000 0.99 0.88sp|P21333|FLNA_HUMAN 80 54 54 73 75 76 63 68 151410000 99843000 81625000 96847000 1.88E+08 254850000 158740000 189660000 1.84 1.08sp|P01024|CO3_HUMAN 7 76 63 82 24 83 69 68 1754800 437500000 227390000 403870000 12066000 817530000 152120000 152540000 1.06 1.07sp|P54296|MYOM2_HUMAN 84 79 75 80 78 64 74 72 527710000 475330000 397520000 432590000 3.85E+08 194390000 360390000 288030000 0.67 0.91sp|Q5VST9|OBSCN_HUMAN 58 36 43 54 49 27 42 43 25384000 18917000 33799000 20633000 33041000 17317000 24378000 24068000 1.00 0.84sp|Q14896|MYPC3_HUMAN 81 79 71 80 75 69 77 75 917570000 931150000 757320000 839530000 9.44E+08 631780000 1.052E+09 917470000 1.03 0.95sp|Q9Y490|TLN1_HUMAN 68 61 41 63 63 69 48 67 95618000 91592000 72088000 86286000 1.13E+08 151620000 93195000 117150000 1.37 1.06sp|P24043|LAMA2_HUMAN 67 66 63 66 59 45 56 59 85824000 78981000 118900000 68198000 94140000 51074000 125930000 72465000 0.98 0.84sp|P12883|MYH7_HUMAN;sp| 74 69 68 75 66 66 73 67 2.04E+09 1.566E+09 1.838E+09 3042800000 1.96E+09 1.787E+09 2.644E+09 1.845E+09 0.97 0.95sp|P35749|MYH11_HUMAN 65 49 35 45 50 43 49 50 69290000 36793000 38859000 26495000 64453000 46216000 66652000 47426000 1.31 0.99sp|Q702N8|XIRP1_HUMAN 46 42 33 56 45 38 41 55 65142000 65074000 44659000 110760000 65981000 47953000 54994000 97807000 0.93 1.01sp|P49327|FAS_HUMAN 7 53 25 14 4 59 64 47 543120 83987000 18504000 1752800 354360 113070000 340260000 63882000 4.94 1.76sp|P35609|ACTN2_HUMAN 64 60 58 64 63 59 60 60 1.268E+09 1.278E+09 970970000 1264800000 1.15E+09 756300000 1.211E+09 930300000 0.85 0.98sp|P35573|GDE_HUMAN 52 44 28 48 44 23 34 50 48687000 31773000 40023000 46956000 50964000 26453000 42478000 56433000 1.05 0.88sp|Q00610|CLH1_HUMAN;sp| 54 46 33 54 56 55 51 58 84732000 56908000 66310000 84364000 1.21E+08 143880000 109010000 115330000 1.67 1.18sp|Q9Y4G6|TLN2_HUMAN 46 43 26 44 39 40 33 39 41499000 41803000 57598000 32082000 46541000 40272000 53893000 42258000 1.06 0.95XX|ALBU_HUMAN|;sp|P02768 34 56 57 57 40 57 59 52 150250000 8.664E+09 8.008E+09 7340200000 3.12E+08 9.386E+09 7.157E+09 2.47E+09 0.80 1.02sp|Q5VTT5|MYOM3_HUMAN 53 50 37 54 45 35 44 47 96031000 84636000 95196000 77917000 96065000 48282000 96419000 79066000 0.90 0.88sp|P18206|VINC_HUMAN 44 44 48 46 47 46 42 49 155040000 133050000 154150000 123820000 1.35E+08 159670000 173090000 161910000 1.11 1.01
Label Free Quantitation--MaxQuant
Label Free Quantitation by Precursor Ion Peak Area
Relative Quantitation of PTM Using Peak Area
RT: 19.96 - 50.19 SM: 7B
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50Time (min)
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Relat
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RT: 31.46AA: 6948813009
RT: 36.22AA: 12379095
RT: 33.66AA: 3238508
RT: 41.54AA: 3445463
RT: 44.32AA: 3944626
RT: 48.01AA: 2336711
RT: 39.67AA: 66098364
RT: 42.77AA: 41752420
RT: 38.84AA: 31864608
RT: 33.82AA: 596224
RT: 35.79AA: 304562
RT: 31.35AA: 126488
RT: 45.02AA: 90790
RT: 49.46AA: 72789
NL: 1.75E8Base Peak m/z= 514.7983-514.8189 F: FTMS + c ESI Full ms [350.00-2000.00] MS ICIS 30138_TRY
NL: 2.39E6Base Peak m/z= 535.8032-535.8246 F: FTMS + c ESI Full ms [350.00-2000.00] MS ICIS 30138_TRY
Sequence m/z Mass Error
Peak Area RT (min)
Total Peak Area
Percentage (%)Theoretical Observed
115GELLEAIKR123 514.80862+ 514.80632+ -4.47 6948813009 31.46 99.40115GELLEAIK(Ace)R123 535.81392+ 535.81362+ -0.56 41739255 42.77 6.99E+09 0.60
115GELLEAIKR123
115GELLEAIK(Ac)R123
(Based on Retention Time and SIC)
emPAI = 10PAI –1PAI=Nobst/Nobsbl
Protein content (mol %) = emPAI/ ∑ (emPAI) x 100
Protein name Conc.
Fmol
emPAI
Protein Content RankConc. emPAI Conc. emPA
IElongation factor 1-a 1 870 9.00 15.04 16.18 2 1a enolase 596 6.50 10.30 11.69 3 2Heat shock protein HSP 90-a 940 6.26 16.25 11.26 1 3Vimentin 336 5.58 5.81 10.03 6 414-3-3 protein 381 4.62 6.58 8.31 5 540 S ribosomal protein S16 456 2.98 7.88 5.36 4 6Pyruvate kinase, M2 isozyme 216 2.06 3.73 3.70 9 740 S ribosomal protein S9 135 1.89 2.33 3.40 12 8GTP-binding nuclear protein RAN 255 1.85 4.41 3.33 8 9ADP, ATP carrier protein, fibroblast isoform
264 1.64 4.56 2.95 7 10
Peripherin 84 1.48 1.45 2.66 18 11Stress-70 protein, mitochondrial precursor
195 1.42 3.37 2.55 11 12
Fructose-bisphosphate aldolase A 210 1.15 3.63 2.07 10 13IgE-binding protein 122 1.15 2.11 2.07 13 14Calreticulin precursor 114 1.15 1.97 2.07 14 1560 S ribosomal protein L11 108 1.15 1.87 2.07 15 1660 S ribosomal protein L17 90 1.00 1.56 1.80 17 17Peroxiredoxin 4 72 0.85 1.24 1.53 20 18Voltage-dependent anion-selective channel protein
54 0.85 0.93 1.53 21 19
T-complex protein 1, e subunit 96 0.81 1.66 1.46 16 20ATP synthase oligomycin sensitivity conferral protein
78 0.70 1.35 1.26 19 21
Phosphate carrier protein, mitochondrial precursor
48 0.55 0.83 0.99 22 22
T-complex protein 1, a subunit B 36 0.52 0.62 0.94 23 23Nucleolar RNA helicase II 30 0.45 0.52 0.81 24 24
Relative Quantitation Using emPAI
Ishihama Y1, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.Mol Cell Proteomics. 2005 Sep;4(9):1265-72
Relative Quantitation Using emPAI
Table Provided Courtesy of Krzycki’s lab