Methods in Cell Biology

Proteomics
z Hong Li, Associate Professor
z Center for Advanced Proteomics Research
(www.umdnj.edu/proweb)
z NJMS Cancer Center
z 973-972-8396
z liho2@umdnj.edu

Where are we? What do we do?

1
Major services at CAPR

¾ Protein identification
¾ Peptide sequencing
¾ Post translational modification (PTM)

¾ Protein purity/mass determination

¾ Proteomics
¾ 2D gel
¾ iTRAQ

¾ Metabolomics

Overview

zProteomics: definition and scopes

zProtein structure and function
zWorkflow and technologies
zApplications
zData interpretation
zAdvanced developments

2
The human proteome

zHuman Genome = 30,000 to 60,000 genes

zHuman Proteome = 300,000 to 1,200,000

protein variants

Functional classification of human proteins (many unknown)

(Lars Juhl Jensen, embl)

Proteomics

3
 Definition and scopes
z Proteomics: systematic & comparative
analysis of protein function
{ Objectives:
z Activities (function)
z Expression
z Post-translational modifications (PTM)
z Localization
z Complex formation

Definition and scopes

{ Techniques:
z Handling protein mixtures: separation
z Mass spectrometry: protein ID & quantification
z Edman sequencing: protein/peptide N-terminal sequencing
z Bioinformatics: changes of protein function in biological
context

z Applications: biology and disease research

{ Molecular event description
(signaling molecules, biomarkers)
{ Finding disease mechanisms and drug targets

4
 Compared with Genomics
z Similarities:
{Static picture of dynamic processes
{High throughput analysis
{Technology-driven
{Computation intensive
z Differences:
{Proteomics: “closer” to activity (function: PTM, location,
turnover, protein complex, enzyme function)
{Protein dynamics and RNA dynamics do not always
correlate

Protein Structure

5
Structural Elements Affecting Function
Post-Translational Modifications

Proteolytic Processing

6
Protein Function

zTwo “broad” types of protein function

{“Catalytic” proteins, including enzymes,
transporters, chaperons, etc.
{“Structural” proteins

Catalytic “Enzymatic” Function

substrates
active site
of enzyme

enzyme

1 Substrates enter
active site in a
specific orientation.

3 Substrates, bonded together, 2 Substrates and active site

leave enzyme; enzyme is change shape, promoting
ready for new set of substrates. reaction between substrates.

7
Structural Proteins are “Functional”

Proteomics – work flow and technologies

z 2-dimensional gel based technologies

z Mass spectrometry for protein identification
z Mass spectrometry for protein quantification

8
2-dimensional gel based technologies

Human Proteome

9
Theoretical pI and Mr map of yeast cell proteins

1000

100
Mr / kDa

10

1
2 4 6 8 10 12 14

Theoretical pI

From: Wildgruber et al. Electrophoresis. 21 (2000) 2610-2616.

2-D Gel Electrophoresis

A powerful technique for protein separation

z 1st dimension: isoelectric focusing (IEF)

- separate proteins based on their isoelectric point (pI) by
using immobilized pH gradient (IPG) gel strips.
z 2nd dimension: SDS PAGE
- separate proteins based on their molecular mass
IEF MW kDa
SDS 200
150
100
75

50

37

25

pI 3 10

10
 2-D image analysis
Control Treated

Difference in Gel Electrophoresis (DIGE)

Protein Protein Protein

Extract 1 Extract 2 Extract 3
Label with Label with Label with
Cy2 Cy3 Cy5

Mix labeled extracts

2D gel separation

Cy2 Cy3 Cy5

Analysis of Differences

11
Protein Expression -
Bioinformatics

Expression Analysis

A B

Excise spot; elute; digest

Extract peptides;
MS analyze
Protein identification

12
 Overview of Mass Spectrometry

Ions formation Separation Detection

ion source mass analyzer ion detector

Relative Intensity %

Inlet
Data System

Sample Introduction
m/z
mass spectrum

Amino Acid Residue Mass: crucial for mass spectrometry-

based protein identification and peptide sequencing

13
 Sample Preparation Cut spots

Gel Electrophoresis

tryptic digestion
cleaves protein at
R and K residues

MS/MS analysis
(peptides are fragmented
in mass spectrometer)

MS Analysis

Peptide Mass Fingerprinting MS/MS Peptide Sequencing Spectra

(PMF) spectrum

Peptide Mass Mapping

14
1000.446
1045.558
1058.51
1060.516
1179.593
1180.588
1234.663
1235.564
1236.56
1308.659
1320.608
1350.694
1353.7
1401.795
1404.783
1417.729
1418.816
1475.757
1488.756
1489.751
1490.765
1493.742
1498.81
1499.816
1707.789
1838.932
1875.97
1901.872
1904 888

15
16
How does the search engine works?

17
 How does the search engine works?

842.509
870.461
882.43
886.448
886.959
898.453
905.657
927.475
937.456

Bovine Serum Albumin peptide 965.484

974.44

2-oxoglutarate dehydrogenase peptide 1003.44

1011.52
1018.75
1026.59
1027.54
1034.45
1042.57
1046.58
1068.42
1072.49
1088.59
1089.61
1150.61
1163.61
1165.59
1198.59
1212.59
1249.6
1271.65
1283.69
1305.69
1394.67
1410.67
1419.67
1435.73
1439.79
1479.78
1502.59
1511.82
1554.64
1567.73
1576.74
1639.92
1724.84
1731.66
1740.82
1747.69
1749.66
1880.91
1907.91
1927.8
1956.95
1959.01
2038.92
2076.06
2100.05
2169.97
2212.1

An example of a spectrum in which not all of the major peaks matched the 2225.12
2239.14
2344.09

primary identification. A second component search reveals the identity of 2359.18

2458.18
2566.18

the other protein. 2807.31

2985.48

18
 Tandem Mass Spectrometry

Tandem Mass Spectrum: An Example

Secondary Fragmentation

Ionized parent peptide

19
 Tandem Mass Spectrometer

detector
ions precursor ions fragment ions

MPSER + PAK + P + AK + + +
mass PAK + P AK + mass AK PA K P+
PAK + fragment
SG… + analyzer PAK + PA + K analyzer
PAK + PA K+
…

de n
ovo
sequ
enci
ng

N- and C-terminal Peptides

es

s
e
tid

tid
ep

ep
lp

lp
a

ina
in
rm

rm
te

te
N-

C-

20
 How Does a Peptide Fragment?

m(b1)=1+m(A1) m(y1)=19+m(A4)
m(b2)=1+m(A1)+m(A2) m(y2)=19+m(A4)+m(A3)
m(b3)=1+m(A1)+m(A2)+m(A3) m(y3)=19+m(A4)+m(A3)+m(A2)

MS/MS Peptide Sequencing

21
 Peptide sequencing by MS/MS

Peptide: [Glu1]-Fibrinopeptide B (EGVNDNEEGFFSAR MW 1569.7844)

MS/MS database search

22
 Database Search of MS/MS Data

1320.585
178.128
195.166
258.121
312.219 1320.584 1320.589 1320.560 1320.583 1320.586
329.204 225.102
233.189
178.117
195.104
200.382
219.491
169.745
185.934
164.046
179.690
436.219 255.192
261.144
223.147
251.210
251.040
282.611
212.659
239.403
205.518
231.364
450.330 382.237 258.162 290.432 246.028 237.767

466.292 vs. 399.228

434.255
436.213
450.349
490.740
506.642
415.711
429.182
401.752
414.771
462.279 484.248 544.779 461.488 445.992
545.365 632.300 485.234 545.888 462.428 446.900
694.343 649.399
662.368
545.357
920.441
613.526
1035.496
519.725
877.180
502.273
847.726
920.455 688.550
809.452
953.450
1057.401
1072.631
1189.576
908.638
1007.703
878.128
973.867
1057.427 843.397 1066.465 1199.773 1016.341 982.215
860.417 1075.395
1066.477
1075.649
1283.684

A-D-L-V-I-P-N-D-F-K

Databases:

Protein databases:

• Swiss-Prot: Curated protein database

high quality, low coverage, modification information

• TrEMBL: translated EMBL cDNA database

medium quality, medium coverage

Nucleotide database:

• Expressed sequence tag (dbEST)

low quality, high coverage

• Genome sequence
high quality, everything included, needs prediction

23
ExPASy

NCBI protein database

24
Protein Databases

zCompiled
GenPept from a variety of resources:
4,297,060
RefSeq
{Translations from annotated 1,616,076
coding
Thirdregions in GenBank and RefSeq. 4,168
Party Annotation
{SwissProt
Swiss Prot 185,800
PIR{PIR (Protein Information Resource)
234,946
PRF{PDB (Protein DataBank– 3D structures)
12,079
PDB 67,416
Total 6,417,545

IPI database

25
 Application 1: Protein Expression Level Changes

Use Proteomics to Investigate Molecular Mechanisms

of Aging and Gender differences in the Heart

Yan et al. J Mol Cell Cardiol. 2004 Nov;37(5):921-9.

Objective

z To reveal aging and gender-associated alterations in

protein expression and function that could explain why
female life expectancy is generally greater than males
and particularly why their cardiovascular risk is less.

26
 Alterations in LV total protein expression in aging monkeys

kDa YM OM YF OF
200 Oxoglutarate dehydrogenase
150
5 1
100
75
1 α -enolase
50 3-Oxoacid CoA transferase 3 2

2
37 Acyl-CoA dehydrogenase
3

4
25
Triosephosphate isomerase 4

5
pH 5 8
Spot No. Proteins
1 3-Oxoacid CoA transferase
2 Acyl-CoA dehydrogenase
3 α-enolase
4 Triosephosphate isomerase
5 Oxoglutarate dehydrogenase

Alterations in mitochondrial protein expression in aging monkeys

YM OM YF OF
kDa

200 1 1 1 1
150 a
100
2 2 2
2
75

50 b
3 3 3 3
37
a c

b
c 4 4 4 4
25

Spot No. Proteins

1 ATP-specific succinyl-CoA synthetase β subunit
2 Pyruvate dehydrogense E1 β subunit
pH 5 8 3 ATP synthase α subunit
4 Acyl-CoA dehydrogenase

27
28
Decreased expression of metabolic enzymes related to energy
production in aging male monkeys
Table 1. Alterations in Protein Expression Related to Metabolic Pathways

Accession Metabolic Detection

Proteins OM vs YM * OF vs YF * OM vs OF *
No. Pathway Methods
3-Oxoacid CoA transferase P55809 Fatty acid oxidation ↑ ↑ ↔ 2DGE(T,M)

Acyl-CoA dehydrogenase P16219 Fatty acid oxidation ↑ ↑ ↔ 2DGE(T)

Alpha-enolase P06733 glycolysis ↓ ↔ ↓ 2DGE(T)

Triosephosphate isomerase P00938 glycolysis ↓ ↔ ↓ 2DGE(T)

P14618
Pyruvate kinase
P14786
glycolysis ↓ ↔ ↓ WB
Pyruvate dehydrogense E1 β
subunit
P11177 Glucose oxidation ↓ ↔ ↓ 2DGE(M)

Oxoglutarate dehydrogenase Q02218 TCA cycle ↓ ↔ ↓ 2DGE(T)

ATP – Specific succinyl-Co A
synthetase β subunit
Q9P2RT TCA cycle ↓ ↔ ↔ 2DGE(M)

ATP synthase α subunit P25705 ETS ↓ ↔ ↓ 2DGE(M), WB

2DGE(T) Two-dimensional gel electrophoresis of total protein extracts

2DGE(M) Two-dimensional gel electrophoresis of isolated mitochondrial proteins
WB Western blotting
↑ indicates increased levels of the protein

↓ indicates decreased levels of the protein

↔ indicates unchanged levels of the protein

* Average change fold was described in Results section of the paper

Application 2: Protein Post-translational Modification (proteolysis)

Proteomic Alterations of Cardiac Troponin T, a Novel Mechanism

for the Transition from Hypertrophy to Heart Failure

29
 Background

z Clinically, the decompensated stage of heart failure (HF) is

usually preceded by chronic, compensatory cardiac
hypertrophy.

z The molecular mechanisms that precipitate the transition

from stable hypertrophy to failure remain largely unknown.

A Proteomic Approach to Determine the Alterations in Protein

Expression in Canine Model

LV tissues: Normal, LVH, LVH/HF

Homogenization
Protein extracts

2D gel electrophoresis (1st: IEF 2nd: SDS-PAGE)

Protein visualization by Sypro Ruby
Gel comparison by computer image analysis

Selected protein spots

Spots excision
In-gel digestion with trypsin
Tryptic peptides
Q-TOF MS/MS
MALDI-TOF MS MALDI-TOF/TOF MS/MS
Peptide masses Peptide sequence

Sequence database search

Protein ID

30
 Alterations of Cardiac Troponin T (cTnT) Expression
in Hypertrophy (LVH) and Heart Failure (LVH/HF)

MW (kDa) Normal LVH LVH/HF

A B C
75
50

37

25

pH 5 8 5 8 5 8
7
7 5 B 5
43 2 C
4 3 2 1
1 6
6
37
8
9 10

25
pH 5 5.5 5 5.5 5 5.5

Identification of cTnT
MALDI mass spectrum of in-gel trypsin digest of spot 5 shown on last slide
Voyager Spec #1 MC[BP = 1943.0, 58237]
1943.0047 5.8E+4
100

90

80

70
1535.8264
60
% Intensity

50 906.4930
2466.1986
40 1945.9842
1629.7696
30
1683.8241
20 1538.8141 2416.1842
10 1155.6182 1408.8022 1622.8152 2469.2021
842.4784 1632.7437 1812.8927
0 0
796.0 1191.8 1587.6 1983.4 2379.2 2775.0
Mass (m/z)

31
 cTnT Degradation in LVH/HF

A C
Voyager Spec #1 MC[BP = 1943.0, 62811] Voyager Spec #1 MC=>BC[BP = 1943.0, 62711] Voyager Spec #1 MC=>NF0.7[BP = 1943.0, 51205]

D
100 4.1E+4 100 5.5E+4 100 7717.5

90 90 90

B
80 80 80

70 70 70

60 60
% Intensity 60

% Intensity
% Intensity
50 50 50

LVH 40

30

20
40

30
40

30

20 20

10 10
10
0 0 0 0
810 868 926 984 1042 1100 0 0 2350 2370 2390 2410 2430 2450
1520 1546 1572 1598 1624 1650
Mass (m/z) Mass (m/z)
Mass (m/z)

Voyager Spec #1 MC[BP = 1943.0, 63027] Voyager Spec #1 MC=>NF0.7=>NF0.7[BP = 1943.0, 62723]

D
Voyager Spec #1 MC[BP = 1943.0, 63027]
100 5.1E+4 100 2375.5
1016.4924 4.3E+4
100
90 90
90
80 80
80
70 70
70
60 60

% Intensity
% Intensity
% Intensity

60

LVH/HF 50

40
50

40
50

40

30 30
30

20 20 20

10 10 10

0 0 0 0 0 0
810 868 926 984 1042 1100 1520 1546 1572 1598 1624 1650 2350 2370 2390 2410 2430 2450
Mass (m/z) Mass (m/z) Mass (m/z)

810 1100 1520 1650 2350 2450

1 225
V L A ID H L N E D Q L R E K A K E L W Q S IY N L E A E K F D IQ E K F K Q Q K
B C
D
Y E IN V L R N R IN D N Q K V S K T R G K A K V T G R W K
A

EL W Q S IYNLEAEK
K E A E L N Y I S Q W LE

MS/MS
Sequencing
Of peptide B

Summary
• Using a proteomic approach, we found
dramatically altered expression of cTnT, an
important regulatory contractile protein, in HF
compared to LVH in our unique canine model.

• Our data indicates that cTnT is degraded near

the C-terminus in HF, but not in LVH.

32
 Data interpretation and advanced application

zPhosphorylation site identification

zProtein complex identification
zProtein isoform problem
zLimitation of current technologies
zEstimation of sample requirement
zMS-based peptide quantification

Posttranslational Modifications (PTM)

Phosphorylation 80
Acetylation 42
Methylation 14
Hydroxy amino acids 16
Acylation
Myristic acid 228
Palmitic acid 256
Prenylation
Farnesol 204
Geranylgeranol 272
Nitrosylation 39 or 45
Oxidation 16
Other oxidation -32 loss of SH
Dityrosine formation
Isoaspartate 1 (+shift of peptide bond)
Glycation variable
Glycoxidation variable
Lipid peroxide adduction variable

33
 Isolating Modified Peptides by Affinity Chromatography Prior
to Tandem Mass Spectrometric Characterization

z Selective enrichment of phosphopeptides with Immobilized Metal Affinity

Chromatography (IMAC)
MS/MS
trypsin IMAC sequencing
Protein Tryptic peptides Purified phosphopeptides Phosphorylation sites

HPLC MALDI-TOF MS

before IMAC before IMAC

after IMAC
after IMAC

4700 Reflector Spec #1[BP = 1591.9, 3337]

1591.94

100
3336.9

Before IMAC
90

(A)
2909.61

80

70
1881.08

60
% Intensity

50
1384.73

40
2061.84

2626.49
2186.17
1267.71

2132.07
1137.56

2424.16

30
2330.57

20

10

0
1100 1490 1880 2270 2660 3050
Mass (m/z)
2061.84

4700 Reflector Spec #1[BP = 2061.8, 8366]

100
8366.3

After IMAC
90

(B) 80

70
2424.16

60
% Intensity

2330.57
1958.18

50

40
2083.81

30
1660.79
1213.07

2909.61

20

10

0
1100 1490 1880 2270 2660 3050
Mass (m/z)

4700 MS/MS Precursor 2062 Spec #1=>NR(30.00)=>NR(50.00)[BP = 1965.3, 921]

H3PO4

100 1964.39 921.4

(C)
90

KDQL E D E T Q Q Q E E pS QF
80

70

60 y8
% Intensity

977.59
50
y8
40 1105.68
y10
y7 1233.74
30
y6 876.53 y11
y5 y13
20
632.43 747.47 1361.84 y12 2061.8
1619.97
10 y4 1490.98 y14
1786.93
0
503.37
276 655 1034 1413 1792 2171
Mass (m/z)

34
Study of Protein-Protein Interactions using Mass
Spectrometry-Based Methods

Affinity purification + Mass spectrometry identification

Isolation of protein complexes by affinity approaches

1D/2D GE or HPLC

Trypsin digestion

Protein identification by mass spectrometry

Affinity approaches for the retrieval of protein

complexes

z Immunoprecipitation
z Epitope-tagging (Myc, HA, Flag, KT3)
z GST-pulldown
z Tandem affinity purification (TAP)

35
2D gel of spliceosome-associated proteins

GST

GST-S14 GST beads

SPF45

GFP-SPF 45

Localization of SPF 45 in nucleus

Meaning of “identifying” a protein

36
Protein isoform problem

Meaning of “quantifying” a protein

37
Limitation of Current Technology

Protein detection on gel

38
How Much Sample is Enough?

39