Quantitative Proteomics LZ 072722

Quantitative Proteomics
Liwen Zhang
Mass Spectrometry and Proteomics Facility
The Ohio State University
Summer Workshop 2022
Campus Chemical Instrument Center

Mass Spec - Proteomics
Quantitative Proteomics
Quantitation 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 (DDA and DIA)

•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)

DIGE (Difference Gel Electrophoresis)
Spot Volume = [spot 1 on treated]/[spot 1 on standard]

Pros and Cons to DIGE
Pros: Cons:
 high sensitivity  300 – 500 µg of total protein is

required for each biological
 linearity of the dyes utilized replicate
 straightforward  Requires high resolution 2D gels

significant reduction of
experiment error  Not ideal for membrane proteins
 High reproducibility  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

TMT ---- Tandem isobaric Mas s Tag for relative and absolute quantitation /
Chemistry of TMT™ 6Plex Reagents
Mass increase---229.1329 Da
Figure Provided Courtesy of Thermo Fisher Scientifics

Chemistry of TMT™ 10/11Plex Reagents
(126C, 127 (N, C), 128 (N,C), 129 (N, C), 130 (N, C), 131 (N, C)
Mass increase---229.1329 Da

Chemistry of TMT Pro™ 16/18Plex Reagents
(126C, 127-133C (N,C), 134N/ (126C, 127-134C (N,C), 135N
Campus Chemical Instrument Center Mass increase---304.2071 Da

Workflow of TMT™ Reagents (up to 18 Samples)

Dry Eye Studies:
4 Groups: Normal, Moderate Dry Eye, Mild Dry Eye, Mixed Dry Eyes
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)
Ratio 126:127:128:129:130:131=1:0.30:0.09:0.25:0.00:1.49
131
100
95
90
Protein Name : Human Cystatin-S
Peptide Sequence:EQTFGGVNYFFDVEVGR
85
80
Relative Ayundance
75
70
65 126
Observed m/z= 1097.02702+
60
55
50
45
40
35
30
25 127 129 Theoretical m/z= 1097.04432+
20
15
10
128
5
0
124 126 128 m/z 130 132 134
1401.32
232.12 331.31 559.25 674.35 821.55 968.30 1131.44 1245.38 1344.23y12
1458.29 1605.40 1706.27 1834.52
y2 y3 y5 y6 y7 y8 y9 y10 y11 y13 y14 y15 y16
99.17 227.94 (129+99) 115.10 147.20 146.75 163.14 113.94 98.85 57.0956.97 147.11 100.87 128.25
Val Glu-Val Asp Phe Phe Tyr Asn Val Gly Gly Phe Thr Gln
9=229 (TMT)+129(E)+1 (H) 792.12

359.16 487.24 588.18 735.20b5 849.14 948.30 1062.54 1225.15 1372.42 1519.43 1634.48 1733.70 1862.61
b1 b2 b3 b4 b6 b7 b8 b9 b10 b11 b12 b13 b14
128.08 100.94 147.02 56.9257.02 99.16 114.24 162.61 147.27 147.01 115.05 99.22 128.91
Gln Thr Phe Gly Gly Val Asn Tyr Phe Phe Asp Val Glu
100 300 500 700 900 m/z 1100 1300 1500 1700 1900

TMT Labeling MASCOT Search
TMT Labeling MASCOT Results

ProteomeDiscoverer TMT Workflow
Processing Workflow
Consesus Workflow

TMT Proteome Discoverer Results
Description Coverage # Peptides# PSMs # Unique P Ratio:127/126 Ratio:128/126 Ratio:129/126 Ratio:130/126 Ratio:131/126
Vimentin OS=Homo sa 92.27468 70 764 68 1.073 1.296 1.159 1.324 0.489
Glyceraldehyde-3-pho 90.44776 33 195 33 1.463 1.884 1.73 1.632 0.548
Actin, cytoplasmic 1 OS 90.4 33 355 18 1.322 1.489 1.411 1.411 0.541
Thioredoxin OS=Homo 88.57143 8 31 8 1.431 1.636 1.647 2.045 0.554
10 kDa heat shock prot 84.31373 11 19 11 1.118 1.143 0.98 0.891 0.395
Ubiquitin carboxyl-ter 84.30493 11 16 11 1.689 1.715 2.015 1.71 0.546
Tubulin beta-4B chain 82.92135 31 169 4 1.521 1.78 1.621 1.438 0.629
Alpha-enolase OS=Hom82.48848 36 161 32 1.505 1.903 1.747 1.596 0.509
Galectin-1 OS=Homo s 82.22222 13 46 13 1.123 1.391 1.291 1.562 0.571
Tubulin beta chain OS= 82.20721 30 176 5 1.502 1.957 1.631 1.439 0.638
Triosephosphate isom 79.37063 22 64 22 1.755 1.741 1.89 1.554 0.532
Annexin A2 OS=Homo 79.05605 34 111 34 1.118 1.403 1.267 1.606 0.563
ATP synthase subunit 76.93762 25 56 25 1.183 1.222 1.258 1.494 0.491
Tubulin alpha-1A chain 76.7184 28 162 3 1.663 2.091 1.901 1.948 0.682
Tubulin alpha-1C chain 76.6147 28 131 4 1.499 1.978 1.723 2.055 0.789
60S acidic ribosomal p 76.52174 6 19 6 1.233 1.452 1.415 1.317 0.45
Calmodulin OS=Homo 76.51007 7 83 7 1.336 1.246 1.288 1.581 0.49
Myosin light polypepti 76.15894 9 35 7 1.362 1.437 1.381 1.434 0.547
Glutathione S-transfer 75.71429 8 35 8 1.514 1.898 1.888 1.951 0.585
Phosphoglycerate mut 75.59055 16 34 16 1.632 1.69 1.745 1.462 0.516
Pyruvate kinase PKM O 75.32957 42 155 42 1.425 1.961 1.711 1.824 0.568
SH3 domain-binding g 74.19355 4 23 4 1.65 1.583 1.776 1.473 0.524
Peptidyl-prolyl cis-tran 71.51515 12 36 12 1.379 1.657 1.466 1.147 0.417
Profilin-1 OS=Homo sa 70.71429 11 38 11 1.389 1.975 1.723 1.954 0.595
Myosin regulatory ligh 70.17544 11 31 8 1.395 1.571 1.543 2.07 0.685
Fructose-bisphosphate 70.05495 25 57 25 1.57 1.613 1.609 1.369 0.532
14-3-3 protein zeta/de 68.57143 19 51 17 1.442 1.806 1.747 2.048 0.62
Case Study—TMT Labeling (3 cell lines, 4 time points, 4 Bio-reps
Pooled-1C
P0-C P6-C P24-C P72-C L0-C L6-C L24-C L72-C K0-C K6-C K24-C K72-C
Pooled-2C
Pooled-1D
P0-D P6-D P24D P72D L0-D L6-D L24-D L72-D K0-D K6-D K24-D K72-D
Pooled-2D
Pooled 1/Pooled2 =3:1
Pooled-1E
P0-E P6-E P24_E P72_E L0-E L6-E L24-E L72-E K0-E K6-E K24-E K72-E
Pooled-2E

Case Study—TMT–TIC, Biological Replicates

Case Study—Protein / Peptide Quan & ID
64% Proteins Fully Quantified 30% Peptides Fully Quantified
Full Quan = Value in every sample, every channel
Note: 97% of identified proteins produced Note: 94% of identified peptides

ratios, but not fully quantified produced ratios, but not fully
(ie. Missing channels in some replicates) quantified

Log10 Abundance
4
2
3
K0 (Rep1, 130C, Sample, F1)

L0 (Rep1, 128N, Sample, F1)
L24 (Rep1, 133C, Sample, F1)
P0 (Rep1, 133N, Sample, F1)
Groups

Pooled2 = 2.26
Pooled1 = 2.69
Pooled1 (Rep1, 134N, Control, F1)

Pooled2 (Rep1, 126, Sample, F1)
2.69-2.26 = 0.431
Unlabeled (Rep1, 132N, Sample, F1)

Pooled1 : Pooled2
10^(0.431) = 2.70 ratio
We expect ratio ~3.0 for

Avg Median log10(Abun)
Avg Median log10(Abun)
2.86 ratio
TMT-C 3x Inj.
Technical Rep
Pooled1 : Pooled2
Case Study—TMT Labeling Method Accuracy (non-normalization)
Case Study—Coefficient of Variance Plots
95% of Proteins have CV < 20%, 68% have CV < 10%
1600 1600 1400

38.49% 36.11%
37.20% 36.17%
1400 1400 1200
32.21% 31.67%
1200 1200
1000
1000 1000
800
19.01%
Count
800
800
17.29% 15.57%
600
600 600
400
400 400 6.53%
6.66% 7.09%
200 2.90% 200 2.61%

200 2.66%
0.64% 0.69% 0.91%
1.35% 1.62% 1.68%
0 0.27% 0 0.24% 0 0.43%
5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40
Proteins - Abundances (Grouped) CVs [%]: P0 Proteins - Abundances (Grouped) CVs [%]: L0 Proteins - Abundances (Grouped) CVs [%]: K0
1400
36.45% 1400 37.02%
34.31%
31.21% 31.66%
1200 31.38%
1200
1000
1000
800 19.15%
800 18.67%
17.62%
600
600
400 7.64% 8.50% 400

6.50%
200 2.85% 4.51%
200 3.17%
0.67% 2.03%
1.57% 0.77%
0 0.48%
5 10 15 20 25 30 35 40 1.04% 0 1.89% 0.32%
5 10 15 20 25 30 35 40
Proteins - Abundances (Grouped) CVs [%]: P72
Proteins - Abundances (Grouped) CVs [%]: K72
Case Study—PCA – TMT Biological Replicates
PCA Protein Abundances (Scaled)
50% of variance
contained by PC Check Loadings
1&2 Plot
Time
Highest PC1 value protein P31327
points
sig up in K (p<.009), very sig down in L (p<8e-7)
stratify
in PC2
L and K sets
stratify by PC1

Case Study—TMT-C Technical Replicate Shared Protein IDs
TMT-C Rep1-3 Protein IDs TMT-C Rep1-3 Full Quan Proteins

Pros and Cons to iTRAQ/TMT
Pros: Cons:
 Requires less total protein than  Mass Spectrometry requirements

DIGE (50 µg or less) are rigorous
 High resolution
 One experiment can determine  Advanced LC chromatography
fold change, protein ID and Post  Long instrument time
High resolution needed for
translational modification

 TMT10plex and above
Up to 18 (27) groups can be


Produces enormous amount of


compared at the same time


data
Samples can come from both
Requiring detailed bioinformatics

tissue or cell culture


analysis
 Expensive

SILAC (Stable isotope labeling by amino acids in cell culture)
Regular Media Lys-depleted Media Heavy Isotopic Labeled Amino

(Lys Light) (Lys Heavy) Acids:
L-Lysine: 13C6, (+6Da), 13C6/15N2
Drug (+8Da), 13C6/15N2/D9 (+17Da),
No Drug 15N /D9 (+11Da), D4 (+4Da)
Treatment 2
L- Arginine: 13C6 (+6Da), 13C6/15N4

(+10Da), 13C6/15N4, D7 (+17Da),
15N /D7 (+11Da)
4
Combination/
Extraction L-Tyrosine: 13C9 (+9Da)
Fractionation/IP
Digestion/
Peptide Identification
1D or 2D LC/MSMS
Change =
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

11353
∆ M =4*11= 44 11396
11309
11351
11279 11393
11200 11300 11400 11500 11600

Mass
17872_1_7 #1858-1959 RT: 22.95-23.41 AV: 13 NL: 5.19E4
T: FTMS + p ESI Full ms [350.00-2000.00]
663.38
100
95
90
672.39
K* =13C6/15N2 labeled Lys
85
80 R* =13C6/15N4 labeled Arg

75
70
65
60 663.88
Relative Abundance
55
50
45
∆ M =10+8= 18Da 672.90
Ratio=Heavy/Light=1:1
40
35
30
25
664.39
20
673.40
15
10 675.40
671.89 674.88
664.89 675.90
5 667.18 673.90
661.29 670.41
662.83 665.35 666.42 668.18 668.89 676.40 678.37 679.39
0
660 662 664 666 668 670 672 674 676 678
m/z
DNIQGITKPAIR DNIQGITK*PAIR*
18Da
18Da MSMS of DNIQGITK*PAIR*
17872_1_7 #1902 RT: 23.14 AV: 1 NL: 1.01E3
T: ITMS + c ESI d Full ms2 663.38@cid35.00 [170.00-1340.00]
873.56 MSMS of DNIQGITKPAIR
685.43
100
95
90
85
80 615.20
75
606.03
703.42
70 456.31
65
855.50
60
466.34
Relative Abundance
55 663.81
654.58
50
45
40 492.54
549.04
35
30 230.07 1001.27
983.57
25
558.07
602.24
20
15 342.89 501.42
511.08 742.11 816.61
798.47
229.90
255.27 342.92 584.37
725.30
10
298.12 411.46
298.04 428.40 696.36
775.75 825.94 983.84 1143.22
1151.56
5 279.94
358.01
784.19 838.40 921.24 1012.89
1027.74 1117.35
1168.47
0
423.46
975.96 1068.81 1195.44
200 300 400 500 600 700 800 900 1000 1100 1200
m/z

17881_13Days #21693-22317 RT: 127.45-130.89 AV: 100 NL: 6.03E5
T: FTMS + c ESI Full ms [350.00-2000.00]
935.19
100
95
90
85
K* =13C6/15N2 labeled Lys

80
75
944.68
70
R* =13C6/15N4 labeled Arg

65
60
Relative Abundance 55
50
45 1012.47
1014.48
40
907.49
35
30 945.93
1082.79
984.88
25
1044.02
20 1041.70
908.80 930.44 987.00 1009.11

15 982.84
1086.07
10 948.17 1047.03 1078.08
914.92 966.48 1001.85 1023.84 1057.00 1064.50
924.47 1032.53
5 957.65 979.78 1091.02
994.55
1095.56
0
920 940 960 980 1000 1020 1040 1060 1080 1100
m/z
Cell Culture Labeled for 4 days
Cell Culture Labeled for 7 days

SILAC Proteome Discoverer

SILAC Proteome Discoverer
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

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 #4
sp|O75083|WDR1_HUMAN 0.73868 0.49787 16.506 0.51647 0.47227 0.49776 0.42986 22.459 16.166 23.143 15.822
sp|P00352|AL1A1_HUMAN 0.56922 0.41693 10.611 0.45488 0.41005 0.42585 0.39198 8.264 9.4221 18.585 36.193
sp|P01903|DRA_HUMAN 0.52018 0.38994 16.517 0.40177 0.45355 0.36485 0.38588 23.371 7.0303 16.759 13.14
sp|P01911|2B1F_HUMAN;sp 0.67564 0.44803 19.404 0.47696 0.51286 0.37077 0.40544 20.528 8.4054 43.195 17.842
sp|P06733|ENOA_HUMAN;s 0.60635 0.42854 16.788 0.47056 0.47441 0.40585 0.39676 15.9 29.008 16.777 16.347
sp|P07437|TBB5_HUMAN;sp 0.61284 0.42665 20.77 0.42878 0.36437 0.33858 0.43109 17.86 62.69 53.347 35.269
sp|P08865|RSSA_HUMAN 0.76633 0.49336 14.318 0.55719 0.50864 0.45037 0.47984 15.75 11.125 4.0767 9.9245
sp|P10412|H14_HUMAN;sp| 0.24239 0.17842 19.985 0.20851 0.19008 0.17122 0.1608 12.001 20.446 18.897 37.116
sp|P14550|AK1A1_HUMAN 0.4772 0.33042 19.756 0.33151 0.45207 0.36085 0.35049 21.881 45.956 28.25 10.978
sp|P14618|KPYM_HUMAN;s 0.69122 0.49247 18.124 0.51133 0.52859 0.47487 0.46953 18.925 75.637 73.303 18.653
sp|P15121|ALDR_HUMAN 0.61551 0.45403 11.468 0.52463 0.49678 0.4284 0.41575 20.116 17.17 12.841 11.27
sp|P22626|ROA2_HUMAN 0.69888 0.48943 17.553 0.48363 0.65696 0.48906 0.58803 18.879 20.255 19.143 23.764
sp|P24534|EF1B_HUMAN 0.5565 0.37586 11.979 0.38499 0.34737 0.30052 0.38896 15.25 18.88 17.484 10.005
sp|P26447|S10A4_HUMAN 0.68422 0.48022 14.233 0.56271 0.54325 0.42162 0.5152 14.47 11.022 8.8936 13.44
sp|P28068|DMB_HUMAN 0.6218 0.43181 11.582 0.46229 0.51408 0.41554 0.41403 17.433 7.709 6.5076 2.8015
sp|P35754|GLRX1_HUMAN 0.52424 0.40059 12.129 0.49731 0.5024 0.31936 0.39223 20.812 16.575 15.126 16.034
sp|P50552|VASP_HUMAN 0.1523 0.11366 10.3 0.12016 0.096602 0.11853 0.097167 18.344 13.171 8.6084 15.248
sp|P52566|GDIR2_HUMAN 0.52698 0.37307 9.9274 0.37773 0.41031 0.35531 0.34494 12.827 15.699 10.656 17.545
sp|P53634|CATC_HUMAN 0.63824 0.44043 18.262 0.52397 0.54729 0.35123 0.40655 23.993 28.347 31.433 12.961
sp|P59998|ARPC4_HUMAN 0.64433 0.44187 7.2632 0.47422 0.41861 0.42377 0.41194 9.033 11.62 2.6723 5.9446
sp|P60866|RS20_HUMAN 0.659 0.47304 13.95 0.59757 0.5963 0.43103 0.44341 9.0905 9.4678 23.935 14.586
sp|P60953|CDC42_HUMAN 0.62253 0.42021 16.084 0.42021 0.36152 0.32089 0.35312 15.44 6.6748 12.261 21.515
sp|P62826|RAN_HUMAN 0.33754 0.23408 9.6979 0.25069 0.28194 0.20331 0.25885 52.033 35.87 10.805 40.448
sp|P67809|YBOX1_HUMAN; 0.60934 0.46068 15.177 0.52521 0.50314 0.40425 0.46776 24.83 1.7427 16.798 2.7154
sp|P68363|TBA1B_HUMAN;s 0.73028 0.48465 8.7514 0.51455 0.49593 0.45062 0.4635 7.8559 14.224 7.6826 5.6296
sp|P84077|ARF1_HUMAN;sp 0.56325 0.41636 6.6611 0.48001 0.43235 0.40981 0.395 11.442 8.5717 0.7576 10.307
sp|Q14019|COTL1_HUMAN 0.58209 0.38527 8.3687 0.41231 0.43285 0.35975 0.38295 6.9712 18.566 15.06 8.6241
sp|Q16555|DPYL2_HUMAN 0.6755 0.4699 17.226 0.56152 0.48393 0.4345 0.43068 22.794 19.963 21.804 2.377
sp|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

In Vivo SILAC Workflow
Cell Reports 2013 3, 552-566DOI: (10.1016/j.celrep.2013.01.003)

SILAC: Pros and Cons
Pros: Cons:
 One experiment can determine  Expensive

fold change, protein ID and Post
translational modification  Special data analysis platform
 Up to several groups can be  Samples come from cell

compared at the same time cultures
 Mixing at the beginning, allow  Time consuming

more sample preparation steps  Only 2-3 Plex can be analyzed
each time
 significant reduction of
experimental error
 High reproducibility


Label-free Quantification (DDA and DIA)
doesn’t use a stable isotope containing compound to
chemically label the protein. It can determine the relative
amount of proteins in two or more biological samples by
comparing peptide peak areas or spectral counting.

Data Dependent Acquisition Workflow
Digestion LC/MS
MS/MS
Peak Area/Ion Intensity Relative Concentration (emPAI)

MASCOT Spectral Counting MASCOT
ProteomeDiscoverer MASCOT/Scaffold
MaxQuant ProteomeDiscoverer
MaxQuant

Label-free quantification by Spectra Counting
Condition 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 Counting
Relative protein quantification is achieved by comparing the number of
identified MS/MS spectra from the same protein in each of multiple
LC/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

Label Free Quantitation by Spectral Counting
# of reports = # of Treatment Conditions X # of Bio-replicates
Scaffold

Spectral Counting Results-Scaffold
Conditions
Bio-Replicates
# of Spectra
P-Value

Spectral Counting Results-Scaffold

Label Free Quantitation by Precursor Ion Peak Area/Intensity
Fundamentally, MS1-based measurements are more accurate and

precise than spectral counting with a better linear dynamic range. This
arises due to a number of weaknesses of spectral counting:
•No direct measurement of peptide ion properties

•The response in terms of spectra per peptide ion is not constant across different
features.
•The linear dynamic range of the method can be limited by saturation effects.
•Dynamic exclusion methods, designed to improve DDA coverage, can also affect
the response.
•There is a stochastic aspect to DDA sampling, hampering reproducibility; DDA
sampling is also biased towards more abundant species, for this reason.

Label Free Quantitation by Precursor Ion Peak Area
112.09 249.10 435.18 522.21 685.28 742.30 855.38 1011.47
b1 b2 b3 b4 b5 b6 b7 b8
pE H W S Y G L R P G* 934.49 748.41 661.38 498.32 441.29 328.21 172.11

y8 y7 y6 y5 y4 y3 y2
LC/MS trace for GnRH @100pg, 10pg, 5pg and 1pg loading amount
RT: 0.00 - 60.00

21.20
100pg NL:
100 591.7933 2.67E8
80 MS Profile for GnRH 592.2940

Base Peak
m/z=
591.7908-
60 @100pg 591.7968
MS
592.7947 32872_GnRH
40
_100pg
20 580 585 590 595 600
20.92
m/z
16.50 22.35 24.07 26.08 29.51 31.37 34.29 35.47 39.29 40.81 44.57 46.32 50.68 53.40 55.19 57.95
0
21.00 NL:
100
20.94 591.7960 1.17E6
Base Peak
Relative Abundance
80 m/z=
592.2967
591.7908-
60
10pg MS Profile for GnRH 591.7968
592.7981 MS
40 @10pg 32872_gnrh_
10pg
20 580 585 590 595 600
m/z
20.70 21.37 24.01 28.30 33.52 34.80 41.06 45.06 52.30 54.10
0
20.97 NL:
100 3.60E5
591.7961
Base Peak
80 m/z=
60
MS Profile for GnRH 592.2973 591.7908-
591.7968
40 5pg @50pg 592.7990

MS
32872_gnrh_
5pg
20
580 585 590 595 600
20.66 23.95 25.29 27.04 29.59 31.79 34.35 m/z 52.55 55.54
0
21.01 NL:
100 1.18E5
591.7955 Base Peak
80 m/z=
592.2970 591.7908-
60 MS Profile for GnRH 591.7968
MS
40 @1pg 32872_gnrh_
1pg 1pg
No MSMS
20
580 585 41.29 590 595 600
20.72 21.29 27.57 27.94 28.40 32.35 34.39 m/z
44.40 52.56
0
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min)

Label Free Quantitation by Precursor Ion Peak Area

Relative Quantitation of PTM Using Peak Area
(Based on Retention Time and SIC)
RT: 19.96 - 50.19 SM: 7B
RT: 31.46 NL: 1.75E8
AA: 6948813009 Base Peak m/z=
100
514.7983-514.8189 F:
90 115GELLEAIKR123 FTMS + c ESI Full ms
[350.00-2000.00] MS
ICIS 30138_TRY
80
70
Relative Abundance
60
50
40
30
20
RT: 33.66
AA: 3238508
10 RT: 36.22 RT: 41.54 RT: 44.32 RT: 48.01
AA: 12379095 AA: 3445463 AA: 3944626 AA: 2336711
0
RT: 39.67 NL: 2.39E6
AA: 66098364 Base Peak m/z=
100
535.8032-535.8246 F:
FTMS + c ESI Full ms
90 [350.00-2000.00] MS
ICIS 30138_TRY
80
RT: 42.77
70 AA: 41752420
RT: 38.84
60
115GELLEAIK(Ac)R123
AA: 31864608
50
40
30
20
10 RT: 31.35 RT: 33.82 RT: 35.79 RT: 45.02 RT: 49.46
AA: 126488 AA: 596224 AA: 304562 AA: 90790 AA: 72789
0
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
Time (min)
Sequence m/z Mass Peak Area RT Total Peak Percentage

Theoretical Observed Error (min) Area (%)
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

Relative Quantitation Using emPAI
(Exponentially modified protein abundance index )
emPAI = 10PAI–1
PAI=Nobst/Nobsbl Protein content (mol %) = emPAI / ∑ (emPAI) x 100
Protein name Conc. emPAI Protein Content Rank
Fmol Conc. emPAI Conc. emPAI
Elongation 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 54 0.85 0.93 1.53 21 19
protein
T-complex protein 1, e subunit 96 0.81 1.66 1.46 16 20
ATP synthase oligomycin sensitivity 78 0.70 1.35 1.26 19 21
conferral protein
Ishihama Y1, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J,
Phosphate carrier protein, mitochondrial 48 0.55 0.83 0.99 22 22
Mann M. Exponentially modified protein abundance index precursor
(emPAI) for estimation of absolute protein amount in T-complex protein 1, a subunit B 36 0.52 0.62 0.94 23 23
proteomics by the number of sequenced peptides per protein. Nucleolar RNA helicase II 30 0.45 0.52 0.81 24 24
Mol Cell Proteomics. 2005 Sep;4(9):1265-72

Data-Dependent Acquisition (DDA)
•Only selected peptides are further fragmented during the second stage of tandem MS
•These selected peptides are chosen within a narrow range of mass-to-charge (m/z) signal intensity
•Typically, the precursors of highest abundance (called the “top N” /”top speed” precursors) are
selected for further analysis
•The top N are typically 10–15 peptides in total (Or peptides detected with given time window)
•MS/MS data acquisition occurs sequentially for each peptide
•The resulting data are used to search an existing database
Pros Cons
 Simpler to set up and analyze  The MS instrument decides on the fly which are
 Lower demand on computational the top N precursors and then fragments them
resources one after the other. This introduces a level of
 Cheaper to run bias.
 Database-dependent algorithms used for  As a result, DDA datasets can contain “gaps”
DDA analysis are generally faster than de where peptides have been identified in some
novo algorithms samples only. Even though some tweaks have
 DDA may be best for targeted analysis been introduced to mitigate this, this remains an
(where the target peptides are in an issue.
existing database) as it offers more  Lower precision and reproducibility than DIA
sensitive quantification than DIA  Low-abundance peptides are under-represented
 Allows relative quantification of peptides
between samples using various chemical
labeling approaches (e.g., SILAC or iTRAQ)

Data Independent Acquisition Workflow
Digestion LC/MS
MS/MS
DIA Data Analysis tools and pipeline

Case Study—A comparison Between DDA and DIA Methods
Samples: 6 human urine samples

Digestion: S-trap
Instrument: Thermo Fusion
Gradient Length: 120min (DDA); 90min (DIA)
Injection Amount: 2µg (DDA); 1µg (DIA)
Figure 1. Venn diagrams depicting the total identifications

(A) and differentially expressed proteins (B) in the DDA
Figure 2. Heatmap generated from DIA
experiments. a total of 1609 proteins were identified and experiments showing the expression of
quantified with 71 unique identifications assigned to AP proteins in the 3 groups. , a total of
(Figure 1A) and 173 differentially expressed proteins in AP 2811 proteins were identified and with
(Figure 1B). over 800 differentially expressed
proteins. The purple cluster represents
>800 differentially expressed proteins
in AP.
Data-Independent Acquisition (DIA)
•All p e p t id e s a r e fr a gm e n t e d a n d a n a lyze d d u r in g t h e second s t a ge o f t a n d e m MS
•Ta n d e m m a s s s p e ct r a a r e a cq u ir e d e it h e r b y fr a gm e n t in g a ll io n s t h a t e n t e r t h e m a s s
s p e ct r o m e t e r a t a give n t im e (ca lle d b r o a d b a n d DIA) o r b y s e q u e n t ia lly fo cu s in g o n a n a r r ow
m / z w in d ow o f p r e cu r s o r s a n d fr a gm e n t in g a ll p r e cu r s o r s d e t e ct e d w it h in t h a t w in d ow
•MS/ MS d a t a a cq u is it io n o ccu r s in p a r a lle l a cr o s s p e p t id e s
•Re s u lt in g MS s p e ct r a a r e h igh ly m u lt ip le xe d (MS2 s p e ct r a )
Pros Cons
 Does not require prior knowledge of the protein  Place a high demand on computational resources
composition of the sample  Data analysis is challenging because of the multiplexed
 Less biased as all peptides are included in the nature of the MS2 spectra
analysis  The robust database-based search methods used for
 Allows greater temporal resolution, which is an DDA cannot be applied directly
advantage for certain analyses (e.g., looking at  Further improvements are required in the tools and
changes in protein expression or post- software used to deconvolute the complex spectra
translational modifications over time within the produced
same tissue)  Fragment ions in MS2 spectra cannot be traced back to
 Can quantify proteins in complex mixtures over their precursors as they can potentially result from
a large dynamic range, thereby overcoming the multiple precursor ions
challenge of undersampling when using DDA  Tends to be more expensive than DDA
 Offers higher precision and better reproducibility  In terms of quantification, DIA has lower sensitivity than
than DDA DDA as the complete spectrum must be scanned,
 Best approach for discovery proteomics as no reducing the acquisition time per data point
assumptions are made (e.g., comparison of large  De novo search algorithms are not as good at
sample cohorts to see differences in protein quantification as database search algorithms, which
expression) can also reduce quantification sensitivity
 DIA data can be retrospectively analyzed with an  Algorithms need to control the false discovery rate
improved algorithm to generate even better among the identified peptides while also identifying as
results many of the real peptides as possible


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Quantitative Proteomics

Summer Workshop 2022

Campus Chemical Instrument Center

proteomics research with the development of quantitation techniques

•Difference Gel Electrophoresis

•Isobaric tag for relative and absolute quantitation – iTRAQ/TMT

•Stable isotope labeling by amino acids in cell culture – SILAC

•Label Free Quantitation (DDA and DIA)

Campus Chemical Instrument Center

Campus Chemical Instrument Center

Spot Volume = [spot 1 on treated]/[spot 1 on standard]

Campus Chemical Instrument Center

 high sensitivity  300 – 500 µg of total protein is

 straightforward  Requires high resolution 2D gels

 High reproducibility  Not ideal for serum type samples

 Some protein spots identify more

Campus Chemical Instrument Center

Chemistry of TMT™ 6Plex Reagents

Figure Provided Courtesy of Thermo Fisher Scientifics

Campus Chemical Instrument Center

Figure Provided Courtesy of Thermo Fisher Scientifics

Campus Chemical Instrument Center

Figure Provided Courtesy of Thermo Fisher Scientifics

Campus Chemical Instrument Center Mass increase---304.2071 Da

Figure Provided Courtesy of Thermo Fisher Scientifics

Campus Chemical Instrument Center

124 126 128 m/z 130 132 134

9=229 (TMT)+129(E)+1 (H) 792.12

Campus Chemical Instrument Center

TMT Labeling MASCOT Results

Campus Chemical Instrument Center

Campus Chemical Instrument Center

Campus Chemical Instrument Center

Campus Chemical Instrument Center

64% Proteins Fully Quantified 30% Peptides Fully Quantified

Full Quan = Value in every sample, every channel

Note: 97% of identified proteins produced Note: 94% of identified peptides

Campus Chemical Instrument Center

K0 (Rep1, 130C, Sample, F1)

P0 (Rep2, 133N, Sample, F2)

Pooled1 (Rep1, 134N, Control, F1)

Unlabeled (Rep1, 132N, Sample, F1)

We expect ratio ~3.0 for

95% of Proteins have CV < 20%, 68% have CV < 10%

1600 1600 1400

200 2.90% 200 2.61%

400 7.64% 8.50% 400

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TMT-C Rep1-3 Protein IDs TMT-C Rep1-3 Full Quan Proteins

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 Requires less total protein than  Mass Spectrometry requirements

 TMT10plex and above

Up to 18 (27) groups can be

Produces enormous amount of

compared at the same time

tissue or cell culture

Campus Chemical Instrument Center

Regular Media Lys-depleted Media Heavy Isotopic Labeled Amino

L- Arginine: 13C6 (+6Da), 13C6/15N4

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

11200 11300 11400 11500 11600

80 R* =13C6/15N4 labeled Arg

Campus Chemical Instrument Center

K* =13C6/15N2 labeled Lys