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 663.38@cid35.00 [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 743.89@cid35.00 [190.00-2000.00]

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

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 998.46@cid35.00 [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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55

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65

70

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100

Rel

ativ

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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 549.61@cid35.00 [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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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 736.41@cid35.00 [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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Rel

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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 834.75@cid35.00 [215.00-2000.00]

400 450 500 550 600 650 700 750 800 850 900m/z

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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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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