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Methods in Cell Biology Proteomics - Hong Ki Et Al Lecture
Methods in Cell Biology Proteomics - Hong Ki Et Al Lecture
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
1
Major services at CAPR
¾ Protein identification
¾ Peptide sequencing
¾ Post translational modification (PTM)
¾ Proteomics
¾ 2D gel
¾ iTRAQ
¾ Metabolomics
Overview
2
The human proteome
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
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
enzyme
1 Substrates enter
active site in a
specific orientation.
7
Structural Proteins are “Functional”
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
50
37
25
pI 3 10
10
2-D image analysis
Control Treated
2D gel separation
Analysis of Differences
11
Protein Expression -
Bioinformatics
Expression Analysis
A B
12
Overview of Mass Spectrometry
Inlet
Data System
Sample Introduction
m/z
mass spectrum
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
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
An example of a spectrum in which not all of the major peaks matched the 2225.12
2239.14
2344.09
18
Tandem Mass Spectrometry
Secondary Fragmentation
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
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)
21
Peptide sequencing by MS/MS
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
A-D-L-V-I-P-N-D-F-K
Databases:
Protein databases:
Nucleotide database:
• Genome sequence
high quality, everything included, needs prediction
23
ExPASy
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
Objective
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
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
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
29
Background
Protein ID
30
Alterations of Cardiac Troponin T (cTnT) Expression
in Hypertrophy (LVH) and Heart Failure (LVH/HF)
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)
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.
32
Data interpretation and advanced application
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
HPLC MALDI-TOF MS
after IMAC
after IMAC
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
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)
(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
1D/2D GE or HPLC
Trypsin digestion
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
SPF45
GFP-SPF 45
36
Protein isoform problem
37
Limitation of Current Technology
38
How Much Sample is Enough?
39