Download Protein Quantitation - The Ohio State University

Quantitative Proteomics
Liwen Zhang
Mass Spectrometry and Proteomics Facility
The Ohio State University
Summer Workshop 2016
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
• 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)
DIGE (Difference Gel Electrophoresis)
Spot Volume = [spot 1 on treated]/[spot 1 on standard]
DIGE (Difference Gel Electrophoresis)
In Gel Digestion, LC/MSMS, MASCOT
Pros and Cons to DIGE
 high sensitivity

 linearity of the dyes utilized
 straightforward
significant reduction of
experiment error
 High reproducibility





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
Isobaric tag for relative and absolute quantitation
Cell Culture + Tissue
Trypsin digestion
Labeling Lys and N‐terminus
Up to 8 samples Fractionation offers more identification
Chemistry of TMT™ Reagents
Figure Provided Courtesy of Thermo Fisher Scientifics Workflow of TMT™ Reagents
Figure Provided Courtesy of Thermo Fisher Scientifics Dry Eye Studies:
4 Groups: Normal, Moderate Dry Eye, Mild Dry Eye, Mixed Dry Eyes
Group
1
2
3
4
5
6
IS
IS 1 (126)
IS 2 (126)
IS 3 (126)
IS 4 (126)
IS 5 (126)
IS 6 (126)
Normal
2004 (127)
2007 (131)
2008 (130)
2063 (129)
2015 (128)
2016 (127)
Mod
2041 (128)
2042 (127)
2065 (131)
2046 (130)
2068 (129)
2067 (128)
Mild
2043 (129)
2040 (128)
2070 (127)
2038 (131)
2071 (130)
2062 (130)
Ratio 126:127:128:129:130:131=1:0.30:0.09:0.25:0.00:1.49
Relative Ayundance
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
124
126
126
129
128
128 m/z 130
232.12 331.31
y2
y3
132
359.16
b1
134
559.25 674.35
y5
y6
99.17 227.94 (129+99) 115.10
Val
Glu-Val
Asp
487.24 588.18
b2
b3
300
500
821.55
y7
147.20
Phe
968.30
y8
146.75
Phe
1401.32
y12
1131.44 1245.381344.23 1458.29
y11
y13
y9
y10
163.14
Tyr
792.12
735.20b5 849.14 948.30 1062.54
b4
b6
b7
b8
128.08 100.94 147.02
Gln
Thr
Phe
100
Protein Name : Human Cystatin-S
Peptide Sequence:EQTFGGVNYFFDVEVGR
Observed m/z= 1097.02702+
Theoretical m/z= 1097.04432+
131
127
Mix
2069 (130)
2066 (129)
2037 (128)
2014 (127)
2036 (131)
2035 (131)
700
56.9257.02 99.16
Gly Gly Val
114.24
Asn
113.94 98.85 57.0956.97 147.11 100.87 128.25
Asn
Val Gly Gly Phe
Thr
Gln
1225.15
b9
162.61
Tyr
900 m/z 1100
1605.40 1706.27 1834.52
y14
y15
y16
1372.42
b10
147.27
Phe
1300
iTraq MASCOT Results
1519.43 1634.481733.70 1862.61
b11
b12
b13
b14
147.01 115.05 99.22 128.91
Phe
Asp
Val
Glu
1500
1700
1900
iTraq Results
DE/NDE (Ratio (# of Detection, p value))
Protein Name
Mild/Normal
Aldehyde dehydrogenase, dimeric NADP-preferring OS=Homo sapiens
(2.41, 6, 0.0030)
Apolipoprotein A-I OS=Homo sapiens
(1.75, 6, 0.0362)
Cystatin-S OS=Homo sapiens
(0.51, 6, 0.0014)
Mod/Normal
Mix/Normal
(1.69, 6, 0.0471)
Function
oxidoreductase activity
transporter activity; protein binding; enzyme regulator activity; lipid binding
(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)
Haptoglobin OS=Homo sapiens
(1.97, 6, 0.0474)
(2.11, 6, 0.0486)
binding; transporter activity; ion binding
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)
Putative lipocalin 1-like protein 1 OS=Homo sapiens
(0.43, 6, 0.0026)
Lipocalin-1 OS=Homo sapiens
(0.33, 6, 0.0030)
Lysozyme C OS=Homo sapiens
(0.33, 6, 0.0019)
(0.28, 6, 0.0031)
Polymeric immunoglobulin receptor OS=Homo sapiens
(0.51, 6, 0.0290)
(0.43, 6, 0.0016)
Prolactin-inducible protein OS=Homo sapiens
(0.36, 6, 0.0004)
(0.38, 6, 0.0008)
Proline-rich protein 1 OS=Homo sapiens
(0.44, 6, 0.0023)
(0.39, 6, 0.0056)
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
(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)
Zinc-alpha-2-glycoprotein OS=Homo sapiens
(0.40, 6, 0.0047)
(0.36, 6, 0.0029)
Serotransferrin OS=Homo sapiens
Zymogen granule protein 16 homolog B OS=Homo sapiens
(0.23, 6, 0.0003)
(0.48, 6, 0.0428)
protein binding; extracellular matrix binding
(0.39, 6, 0.0002)
(0.50, 6, 0.0119)
binding; transporter activity
(0.32, 6, 0.0003)
(0.43, 6, 0.0188)
binding; transporter activity; protein binding; enzyme regulator activity
catalytic activity; hydrolase activity; protein binding
protein binding
(0.58, 6, 0.0409)
protein binding
enzyme regulator activity
transporter activity; steroid binding; vitamin binding;: protein binding
(0.59, 6, 0.0502)
(0.44, 6, 0.0383)
lipid binding; carboxylic acid binding; hydrolase activity; transporter activity
carbohydrate binding
Pros and Cons to iTRAQ




Requires less total protein than
DIGE (50 µg or less)
•
High resolution
Advanced LC chromatography
Long instrument time
PQD/HCD needed on IT instrument
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 rigorous
•
•
•
Produces enormous
amount of data
Requiring detailed
bioinformatic analysis
Expensive
SILAC (Stable isotope labeling by amino acids in cell culture)
Lys-depleted Media
(Lys Heavy)
Regular Media
(Lys Light)
Drug
Treatment
No Drug
Heavy Isotopic Labeled Amino
Acids:
L-Lysine: 13C6, (+6Da), 13C6/15N2
(+8Da), 13C6/15N2/D9 (+17Da),
15N /D9 (+11Da), D4 (+4Da)
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
11393
11279
11200
11300
11400
Mass
11500
11600
17881_13Days #21693-22317
RT: 127.45-130.89
T: FTMS + c ESI Full ms [350.00-2000.00]
935.19
100
AV: 100
NL: 6.03E5
95
90
85
K* =13C6/15N2 labeled Lys
R* =13C6/15N4 labeled Arg
80
75
944.68
70
R
e
la
t
iv
e
A
b
u
n
d
a
n
c
e
65
60
55
50
1012.47
45
1014.48
40
907.49
35
945.93
30
1082.79
984.88
25
1044.02
20
1041.70
930.44
908.80
15
982.84
1009.11
987.00
1086.07
10
914.92
966.48
957.65
1001.85
979.78
1023.84
1078.08
1047.03
1057.00
948.17
924.47
5
1032.53
1064.50
1091.02
1095.56
994.55
0
920
940
960
980
1000
m/z
1020
1040
1060
1080
1100
Cell Culture Labeled for 4 days
Cell Culture Labeled for 7 days
17872_1_7 #1858-1959
RT: 22.95-23.41
T: FTMS + p ESI Full ms [350.00-2000.00]
663.38
100
AV: 13
NL: 5.19E4
95
K* =13C6/15N2 labeled Lys
R* =13C6/15N4 labeled Arg
672.39
90
85
80
75
70
R
e
la
tiv
e
A
b
u
n
d
a
n
c
e
65
60
663.88
55
∆ M =10+8= 18Da
50
45
Ratio=Heavy/Light=1:1
672.90
40
35
30
25
664.39
20
673.40
15
10
671.89
664.89
5
667.18
661.29
0
660
662.83
662
665.35
664
666.42
666
673.90
670.41
668.18
675.40
674.88
675.90
676.40
668.89
668
670
m/z
672
674
678.37
676
679.39
678
DNIQGITK*PAIR*
DNIQGITKPAIR
18Da
18Da
MSMS of DNIQGITK*PAIR*
MSMS of DNIQGITKPAIR
873.56
17872_1_7 #1902
RT: 23.14
AV: 1
NL: 1.01E3
T: ITMS + c ESI d Full ms2 [email protected] [170.00-1340.00]
685.43
100
95
90
85
615.20
80
75
606.03
70
703.42
456.31
65
R
e
la
tiv
eA
b
u
n
d
a
n
c
e
855.50
60
466.34
663.81
654.58
55
50
45
492.54
40
549.04
35
30
230.07
1001.27
983.57
25
558.07
602.24
20
501.42
511.08
342.89
15
255.27
229.90
10
298.12
298.04
5
0
200
279.94
300
342.92
725.30
696.36
428.40
358.01
423.46
400
500
816.61
798.47
584.37
411.46
742.11
600
700
m/z
775.75
784.19
825.94
838.40
800
983.84
975.96
921.24
900
1012.89
1027.74
1068.81
1000
1151.56
1143.22
1168.47
1195.44
1117.35
1100
1200
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



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

 Expensive
 Special data analysis
platform
 Samples come from cell
cultures
 Time consuming
Label-free quantification
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.
LC/MSMS
#
1
b
Seq.
y
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
GLLEDLGYDVVVK
CASP4_MOUSE
Caspase-4
OS=Mus musculus
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
147.1128
K
Data Dependent Acquisition and Dynamic Exclusion
Data Dependent Top5 Method
30143_WT_LEG #14457-14668
RT: 88.19-89.24
T: FTMS + c ESI Full ms [350.00-2000.00]
AV: 19
1
NL: 3.22E5
743.89
100
3
95
90
85
2
649.66
4
80
5
75
70
6
65
R
e
la
tiv
e
A
b
u
n
d
a
n
c
e
499.30
997.60
736.42
729.64
60
7
584.62
55
758.15
50
45
991.52
40
8
35
30
562.36
643.41
788.92
25
445.12
958.96
20
1007.50
508.31
15
601.57
724.35
538.31
450
500
550
600
2
898.13
90
750
80
95
30143_WT_LEG #14306 RT: 87.35 AV: 1 NL: 7.47E2
T: ITMS + c ESI d Full ms2 [email protected] [260.00-2000.00]
962.47
776.52
95
90
85
80
918.39
926.34
468.12
525.26
693.22
25
660.95
20
617.69
15
766.40
816.38
850.10
50
1019.05
550
600
650
700
25
85
819.15
80
800
m/z
850
900
950
1000
1050
1100
624.99
10
738.98
5
15
574.64
945.07
409.65
850.52
420.13
501.05
722.80
693.97
10
468.92
418.44
0
400
601.35
511.27
450
500
767.95
668.46
0
400
833.56
540.92
724.31
611.22
950.34
497.15
898.17 500
450
737.84
550
963.19
809.47
793.33
1037.29
600
650
750
1066.54 700
1167.83
1136.33
1022.35
800
m/z
1196.35
1101.94
1050
1100
1193.31
1150
1200
600
650
700
750
800
m/z
850
900
746.46
50
660.79
45
711.89
822.64
842.66
489.08
950
1000
1050
1100
1150
1200
25
641.63
807.75
674.05
854.16
725.63
799.31
20
824.14
761.20
932.45
55
869.10
30
648.24
60
566.84
540.10
881.19
599.46
15
50
1019.31
1100.22
873.75
45
850
40
900
950
1000
1132.30
644.32
1100
1050
10
580.62
526.61
35
579.50
550
706.20
35
1001.25
466.65
1200
878.39
60
55
70
715.31
651.39
65
20
696.97
75
510.16
566.39
1150
15
617.37
1002.30
40
557.39
20
5
90
45
25
750
417.30
65
95
515.34
50
40
30
500
75
70
100
35
1092.28
450
1000
MS Cycle: 0.06S
LC Peak Width >12S
817.42
80
60
55
30
1120.23 1167.30
35
762.93
85
30143_WT_LEG #14467 RT: 88.20 AV: 1 NL: 4.03E3
T: ITMS + c ESI d Full ms2 [email protected] [190.00-1485.00]
1048.37
45
40
424.27
454.82
5
0
400
869.18
65
734.69
60
55
Relative Abundance
30
Relative Abundance
35
950
95
65
70
40
900
90
70
75
Relative Abundance
638.90
560.70
878.32
80
589.17
50
45
679.63
75
85
55
Relative Abundance
60
907.18
90
100
70
850
30143_WT_LEG #14359 RT: 87.63 AV: 1 NL: 3.35E2
T: ITMS + c ESI d Full ms2 [email protected] [215.00-2000.00]
546.11
100
4
991.16
65
800
m/z
5
30143_WT_LEG #14360 RT: 87.64 AV: 1 NL: 1.09E3
T: ITMS + c ESI d Full ms2 [email protected] [140.00-1660.00]
622.91
100
85
Relative Abundance
700
3
712.51
95
10
650
1154.23
MSMS
1
75
1163.58
1077.51
30143_WT_LEG #14664 RT: 89.25 AV: 1 NL: 1.39E3
T: ITMS + c ESI d Full ms2 [email protected] [190.00-2000.00]
100
912.40
876.43
817.43
837.38
668.66
683.33
639.64
480.61
5
0
400
1063.19
1044.18
947.46
417.92
10
1150
5
1200
886.21
432.28
0 874.36
400
711.34
450
500
550
600
1158.45
650
m/z
700
750
800
850
Dynamic Exclusion
900
30
25
1097.28
727.35
470.06
984.16
20
15
442.20
598.03
487.98
10
693.59
509.03
580.22
562.69
500
550
5
424.11
0
400
450
805.93
622.06
600
795.91
650
700
750
800
m/z
868.80
850
919.63
900
1080.75
1068.94
967.13
950
1000
1050
1118.91
1100
1150
1193.02
1200
Make MSMS for the lower abundant ions possible!!
Proteomics Work Flow
Digestion
LC/MS
MS/MS
Peak Area
MASCOT
ProteomeDiscoverer
Spectral Counting
MASCOT/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 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
Spectral Counting Results-Scaffold
Spectral Counting Results
Label Free Quantitation by Precursor Ion Peak Area
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
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
1D SDS PAGE Fractionation LC/MSMS Workflow
Same Volume
1
In Gel Digestion
LC/MSMS
2
In Gel Digestion
LC/MSMS
3
In Gel Digestion
LC/MSMS
4
5
6
7
In Gel Digestion
In Gel Digestion
In Gel Digestion
In Gel Digestion
LC/MSMS
LC/MSMS
LC/MSMS
LC/MSMS
8
In Gel Digestion
LC/MSMS
9
In Gel Digestion
LC/MSMS
10
In Gel Digestion
LC/MSMS
11
In Gel Digestion
LC/MSMS
>1800 Proteins identified
MASCOT
Search
Global Digestion: 920 Proteins
SCX‐LC/MSMS: 981 Proteins
1D SDS‐LC/MSMS: >1800 Proteins
Multi-dimensional Separation
pH 2.6
pH 10.0
$60/fraction on the orbitrap XL
$75/fraction on the orbitrap
Fusion
F
1
10
15
20
25
30
35
40
45
Time (min)
50
55
60
65
70
RT: 0.00 - 90.00
45.29
100
F2
90
80
45.96
RelativeAbundance
70
60
50
10
15
20
25
30
35
40
Time (min)
43.94
38.87
40
45
50
55
60
54.28
30
61.83
20
30.07
29.06
25.19
22.86
10
4.27
0
0
10
20
30
F1
5
65.46
68.90
71.05
21.37
40
50
Time (min)
60
70
87.65
80
Fractionation (F)
9
25
Gradient separation
30
35
40
Time (min)
45
50
55
60
65
Slide courtesy of Nilini S. Ranbaduge
37
Human HEK293 Cell 2D LC/MSMS 4ug Loading; 3 replicates 152
2430
Sequest HT (D2)
Sequest HT (D2)
183
2938
Sequest HT (E2)
Sequest HT (E2)
223
4644
15492
2975
3690
227
2187
138
2577
157
Sequest HT (F2)
Sequest HT (F2)
Total
Label
3537 Sequest HT (D2)
Sequest HT (E2)
3488
3497 Sequest HT (F2)
3127 Sequest HT (D2)/(E2)
3113 Sequest HT (E2)/(F2)
3202 Sequest HT (D2)/(F2)
2975 Sequest HT (D2)/(E2)/(F2)
4055
Description
Identified by: Sequest HT (D2)
Identified by: Sequest HT (E2)
Identified by: Sequest HT (F2)
Identified by: Sequest HT (D2)/ (E2)
Identified by: Sequest HT (E2)/ (F2)
Identified by: Sequest HT (D2)/(F2)
Identified by: Sequest HT (D2)/(E2)/(F2)
Total
Label
24550 Sequest HT (D2)
24753 Sequest HT (E2)
23946 Sequest HT (F2)
17922 Sequest HT (D2)/(E2)
17679 Sequest HT (E2)/(F2)
19182 Sequest HT (D2)/(F2)
15492 Sequest HT (D2)/(E2)/(F2)
33958
Description
Identified by: Sequest HT (D2)
Identified by: Sequest HT (E2)
Identified by: Sequest HT (F2)
Identified by: Sequest HT (D2)/ (E2)
Identified by: Sequest HT (E2)/ (F2)
Identified by: Sequest HT (D2)/(F2)
Identified by: Sequest HT (D2)/(E2)/(F2)
Relative Quantitation of PTM Using Peak Area
(Based on Retention Time and SIC)
RT: 19.96 - 50.19
SM: 7B
115GELLEAIKR123
RT: 31.46
AA: 6948813009
100
NL: 1.75E8
Base Peak m/z=
514.7983-514.8189 F:
FTMS + c ESI Full ms
[350.00-2000.00] MS
ICIS 30138_TRY
90
Relative Abundance
80
70
60
50
40
30
20
RT: 33.66
AA: 3238508
RT: 36.22
AA: 12379095
10
0
RT: 41.54
RT: 44.32
AA: 3445463 AA: 3944626
RT: 39.67
AA: 66098364
100
RT: 48.01
AA: 2336711
NL: 2.39E6
Base Peak m/z=
535.8032-535.8246 F:
FTMS + c ESI Full ms
[350.00-2000.00] MS
ICIS 30138_TRY
90
80
115GELLEAIK(Ac)R123
RT: 42.77
AA: 41752420
70
RT: 38.84
AA: 31864608
60
50
40
30
20
10
RT: 33.82
RT: 31.35
AA: 126488 AA: 596224
0
20
22
24
26
28
Sequence
115GELLEAIKR123
115GELLEAIK(Ace)R123
30
32
RT: 35.79
AA: 304562
34
36
Time (min)
m/z
38
RT: 45.02
AA: 90790
40
Theoretical
514.80862+
Observed
514.80632+
Mass Error
‐4.47
535.81392+
535.81362+
‐0.56
42
44
Peak Area
46
RT: 49.46
AA: 72789
48
50
Total Peak Area
6948813009
RT (min)
31.46
Percentage (%)
99.40
41739255
42.77
6.99E+09
0.60
Relative Quantitation Using emPAI
emPAI = 10PAI -1
Protein content (mol %) = emPAI/ ∑ (emPAI) x 100
Protein name
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
Conc
.
Fmol
870
596
940
336
381
456
216
135
255
264
9.00
6.50
6.26
5.58
4.62
2.98
2.06
1.89
1.85
1.64
15.04
10.30
16.25
5.81
6.58
7.88
3.73
2.33
4.41
4.56
16.18
11.69
11.26
10.03
8.31
5.36
3.70
3.40
3.33
2.95
Rank
Conc. emPA
I
2
1
3
2
1
3
6
4
5
5
4
6
9
7
12
8
8
9
7
10
84
195
1.48
1.42
1.45
3.37
2.66
2.55
18
11
11
12
210
122
114
108
90
72
54
1.15
1.15
1.15
1.15
1.00
0.85
0.85
3.63
2.11
1.97
1.87
1.56
1.24
0.93
2.07
2.07
2.07
2.07
1.80
1.53
1.53
10
13
14
15
17
20
21
13
14
15
16
17
18
19
T-complex protein 1, e subunit
ATP synthase oligomycin sensitivity
conferral protein
96
78
0.81
0.70
1.66
1.35
1.46
1.26
16
19
20
21
Phosphate carrier protein, mitochondrial
precursor
48
0.55
0.83
0.99
22
22
T-complex protein 1, a subunit B
Nucleolar RNA helicase II
36
30
0.52
0.45
0.62
0.52
0.94
0.81
23
24
23
24
Elongation factor 1-a 1
a enolase
Heat shock protein HSP 90-a
Vimentin
14-3-3 protein
40 S ribosomal protein S16
Pyruvate kinase, M2 isozyme
40 S ribosomal protein S9
GTP-binding nuclear protein RAN
ADP, ATP carrier protein, fibroblast
isoform
Peripherin
Stress-70 protein, mitochondrial
precursor
Fructose-bisphosphate aldolase A
IgE-binding protein
Calreticulin precursor
60 S ribosomal protein L11
60 S ribosomal protein L17
Peroxiredoxin 4
Voltage-dependent anion-selective
channel protein
emPA
I
Protein Content
Conc. emPAI