Quantitative Proteomics

Liwen Zhang

Mass Spectrometry and Proteomics Facility

The Ohio State University

Summer Workshop 2016

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• 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)

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

Chemistry of TMT™ Reagents

Figure Provided Courtesy of Thermo Fisher Scientifics 

Figure Provided Courtesy of Thermo Fisher Scientifics 

Workflow of TMT™ Reagents

Group IS Normal Mod Mild Mix

1 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

1401.32y121458.29y13

1605.40y14

1706.27y15

1834.52y16

487.24b2

588.18b3

792.12b5 849.14

b6948.30b7

1062.54b8

1225.15b9

1372.42b10

1519.43b11

1634.48b12

1733.70b13

1862.61b14

735.20b4

359.16b1

Peptide Sequence:EQTFGGVNYFFDVEVGRProtein Name : Human Cystatin-S

Observed m/z= 1097.02702+

Theoretical m/z= 1097.04432+

124 126 128 130 132 134m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

yund

ance 126

127 129

128

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

Pros and Cons to iTRAQ

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 Incorporation

S 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 Abundance

935.19

944.68

1012.47

907.49

1082.79984.88

1044.02

945.93

982.84908.80

1086.07

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

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 Abundance

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

Relative Abundance

685.43

606.03456.31

855.50

654.58

492.54549.04

983.57

742.11511.08798.47

229.90 342.92 584.37

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

1001.27230.07

558.07

602.24501.42 816.61342.89

725.301143.22

255.27

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 0.379

O00299 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

Pros and Cons to iTRAQ

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 13

2 171.1128 L 1362.7515 12

3 284.1969 L 1249.6674 11

4 413.2395 E 1136.5834 10

5 528.2664 D 1007.5408 9

6 641.3505 L 892.5138 8

7 698.3719 G 779.4298 7

8 861.4353 Y 722.4083 6

9 976.4622 D 559.3450 5

10 1075.5306 V 444.3180 4

11 1174.5990 V 345.2496 3

12 1273.6674 V 246.1812 2

13 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 Abundance

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

3

2

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

Re

lativ

e A

bu

nda

nce

898.13712.51

991.16

962.47776.52

918.39

926.34

878.32589.17

638.90560.70

869.18

734.69

525.26 693.22468.12 766.40 816.38

850.10660.95 1019.05

1048.37617.69

1167.301120.23424.27

454.82

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

622.91

679.63

515.34

819.15

557.39

510.16

715.31651.39566.39 1001.25

466.65574.64

932.45

540.92409.65 724.31501.05

420.13 611.22 809.47 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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

1002.30

648.24

761.20 824.14

644.32

874.36711.34

1158.45

1097.28727.35984.16470.06

442.20 598.03487.98

693.59805.93

580.22509.03 622.06562.69 919.63795.91 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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

546.11

762.93817.42

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

4

5

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 AreaMASCOT

ProteomeDiscoverer

Spectral CountingMASCOT/Scaffold

ProteomeDiscoverer

Relative Concentration (emPAI)MASCOT

Label-free quantification by Spectra Counting

Condition A Condition B

Trypsin Digestion LC/MSMS Analysis

Label Free Quantitation by Spectral Counting

Relative 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

Label Free Quantitation by Precursor Ion Peak Area

Description Peak Area # of Spectra

Run 1 Run 2 Run 3 Run 1 Run 2 Run 3

Histone H4 OS=Homo sapiens GN=HIST1H4A PE=1 SV=2 ‐ [H4_HUMAN] 5.99E+10 4.24E+10 5.46E+10 689 546 627

Histone H2B type 1‐K OS=Homo sapiens GN=HIST1H2BK PE=1 SV=3 ‐ [H2B1K_HUMAN] 3.5E+10 7.82E+09 1.06E+10 424 376 418

Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 ‐ [ACTB_HUMAN] 1.64E+10 1.12E+10 1.47E+10 632 507 637

Beta‐actin‐like protein 2 OS=Homo sapiens GN=ACTBL2 PE=1 SV=2 ‐ [ACTBL_HUMAN] 1.55E+10 4.74E+09 7.22E+09 115 90 119

Actin, alpha cardiac muscle 1 OS=Homo sapiens GN=ACTC1 PE=1 SV=1 ‐ [ACTC_HUMAN] 1.34E+10 9.02E+09 1.27E+10 295 223 266

Actin, alpha skeletal muscle OS=Homo sapiens GN=ACTA1 PE=1 SV=1 ‐ [ACTS_HUMAN] 1.34E+10 9.02E+09 1.27E+10 293 223 266

Histone H2A.Z OS=Homo sapiens GN=H2AFZ PE=1 SV=2 ‐ [H2AZ_HUMAN] 1.18E+10 2.7E+10 3.2E+10 354 296 347

Histone H2A type 1‐D OS=Homo sapiens GN=HIST1H2AD PE=1 SV=2 ‐ [H2A1D_HUMAN] 1.13E+10 2.73E+10 3.16E+10 482 443 477

Histone H2A type 2‐A OS=Homo sapiens GN=HIST2H2AA3 PE=1 SV=3 ‐ [H2A2A_HUMAN] 1.13E+10 2.73E+10 3.16E+10 474 438 474

>XX|RS27A_HUMAN| 1.09E+10 5.32E+09 1.02E+10 89 75 83

Heterogeneous nuclear ribonucleoproteins A2/B1 OS=Homo sapiens GN=HNRNPA2B1 PE=1 

SV=2 ‐ [ROA2_HUMAN] 9.76E+09 6.27E+09 8.81E+09 369 253 280

Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 ‐ [VIME_HUMAN] 8.02E+09 5.64E+09 8.38E+09 1056 934 993

Complement component 1 Q subcomponent‐binding protein, mitochondrial OS=Homo 

sapiens GN=C1QBP PE=1 SV=1 ‐ [C1QBP_HUMAN] 6.16E+09 3.52E+09 5.57E+09 303 240 280

>XX|TRYP_PIG| 5.32E+09 1.56E+10 6.16E+09 248 228 239

60 kDa heat shock protein, mitochondrial OS=Homo sapiens GN=HSPD1 PE=1 SV=2 ‐

[CH60_HUMAN] 5.25E+09 3.52E+09 4.87E+09 762 682 789

ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATP5B PE=1 SV=3 ‐

[ATPB_HUMAN] 5.08E+09 3.25E+09 4.61E+09 420 316 427

Label Free Quantitation by Precursor Ion Peak Area

1

2

3

4567

8

9

10

11

In Gel Digestion

In Gel Digestion

In Gel Digestion

In Gel DigestionIn Gel DigestionIn Gel DigestionIn Gel Digestion

In Gel Digestion

In Gel Digestion

In Gel Digestion

In Gel Digestion

Same Volume

LC/MSMS

LC/MSMS

LC/MSMS

LC/MSMSLC/MSMSLC/MSMSLC/MSMS

LC/MSMS

LC/MSMS

LC/MSMS

LC/MSMS

MASCOT Search

1D SDS PAGE Fractionation LC/MSMS Workflow

>1800 Proteins identifiedGlobal Digestion:     920 ProteinsSCX‐LC/MSMS:         981 Proteins1D SDS‐LC/MSMS: >1800 Proteins

Multi-dimensional Separation

Fractionation (F)

pH 10.0

Gradient separation

pH 2.6

37

RT: 0.00 - 90.00

0 10 20 30 40 50 60 70 80 9Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abu

ndanc

e

45.29

45.96

43.9438.87

54.28

61.83

30.0765.4629.06

25.1922.86 68.90

71.054.27 21.37 87.65

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

10 15 20 25 30 35 40 45 50 55 60Time (min)

25 30 35 40 45 50 55 60 65Time (min)

F1

F2

F15

Slide courtesy of Nilini S. Ranbaduge

$60/fraction on the orbitrap XL$75/fraction on the orbitrapFusion

Sequest HT (D2)

2938

Sequest HT (E2)

4644

Sequest HT (F2)

2577

2430

2187

3690

15492

Sequest HT (D2)183

Sequest HT (E2)

223

Sequest HT (F2)

157

152

138

227

2975

Total Label Description

3537 Sequest HT (D2) Identified by: Sequest HT (D2)

3488 Sequest HT (E2) Identified by: Sequest HT (E2)

3497 Sequest HT (F2) Identified by: Sequest HT (F2)

3127 Sequest HT (D2)/(E2) Identified by: Sequest HT (D2)/ (E2)

3113 Sequest HT (E2)/(F2) Identified by: Sequest HT (E2)/ (F2)

3202 Sequest HT (D2)/(F2) Identified by: Sequest HT (D2)/(F2)

2975 Sequest HT (D2)/(E2)/(F2) Identified by: Sequest HT (D2)/(E2)/(F2)

4055

Total Label Description

24550 Sequest HT (D2) Identified by: Sequest HT (D2)

24753 Sequest HT (E2) Identified by: Sequest HT (E2)

23946 Sequest HT (F2) Identified by: Sequest HT (F2)

17922 Sequest HT (D2)/(E2) Identified by: Sequest HT (D2)/ (E2)

17679 Sequest HT (E2)/(F2) Identified by: Sequest HT (E2)/ (F2)

19182 Sequest HT (D2)/(F2) Identified by: Sequest HT (D2)/(F2)

15492 Sequest HT (D2)/(E2)/(F2) Identified by: Sequest HT (D2)/(E2)/(F2)

33958

Human HEK293 Cell 2D LC/MSMS 4ug Loading; 3 replicates 

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)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bunda

nce

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 99.40

115GELLEAIK(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 -1 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 1

a enolase 596 6.50 10.30 11.69 3 2

Heat shock protein HSP 90-a 940 6.26 16.25 11.26 1 3

Vimentin 336 5.58 5.81 10.03 6 4

14-3-3 protein 381 4.62 6.58 8.31 5 5

40 S ribosomal protein S16 456 2.98 7.88 5.36 4 6

Pyruvate kinase, M2 isozyme 216 2.06 3.73 3.70 9 7

40 S ribosomal protein S9 135 1.89 2.33 3.40 12 8

GTP-binding nuclear protein RAN 255 1.85 4.41 3.33 8 9

ADP, ATP carrier protein, fibroblast isoform

264 1.64 4.56 2.95 7 10

Peripherin 84 1.48 1.45 2.66 18 11

Stress-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 13

IgE-binding protein 122 1.15 2.11 2.07 13 14

Calreticulin precursor 114 1.15 1.97 2.07 14 15

60 S ribosomal protein L11 108 1.15 1.87 2.07 15 16

60 S ribosomal protein L17 90 1.00 1.56 1.80 17 17

Peroxiredoxin 4 72 0.85 1.24 1.53 20 18

Voltage-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 20

ATP 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 23

Nucleolar 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