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8/14/2019 Methods in Cell Biology Proteomics _ Hong Ki Et Al Lecture
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Methods in Cell Biology
ProteomicsHong Li, Associate Professor Center for Advanced Proteomics Research(www.umdnj.edu/proweb)NJMS Cancer Center 973-972-8396
liho2@umdnj.edu
Where are we? What do we do?
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Major services at CAPR
Protein identificationPeptide sequencingPost translational modification (PTM)
Protein purity/mass determination
Proteomics2D gel
iTRAQ
Metabolomics
Overview
Proteomics: definition and scopesProtein structure and functionWorkflow and technologies
ApplicationsData interpretationAdvanced developments
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Human Genome = 30,000 to 60,000 genes
Human Proteome = 300,000 to 1,200,000protein variants
The human proteome
Functional classification of human proteins (many unknown)(Lars Juhl Jensen, embl)
Proteomics
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Definition and scopesProteomics: systematic & comparativeanalysis of protein function
Objectives:Activities (function)ExpressionPost-translational modifications (PTM)LocalizationComplex formation
Definition and scopes
Techniques:Handling protein mixtures: separationMass spectrometry: protein ID & quantification
Edman sequencing: protein/peptide N-terminal sequencingBioinformatics: changes of protein function in biologicalcontext
Applications: biology and disease researchMolecular event description(signaling molecules, biomarkers)Finding disease mechanisms and drug targets
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Compared with Genomics
Similarities:Static picture of dynamic processesHigh throughput analysisTechnology-drivenComputation intensive
Differences:Proteomics: closer to activity (function: PTM, location,turnover, protein complex, enzyme function)Protein dynamics and RNA dynamics do not always
correlate
Protein Structure
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Structural Elements Affecting FunctionPost-Translational Modifications
Proteolytic Processing
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Protein Function
Two broad types of protein functionCatalytic proteins, including enzymes,transporters, chaperons, etc.Structural proteins
Catalytic Enzymatic Function
substrates
3 Substrates, bonded together,leave enzyme; enzyme isready for new set of substrates.
active siteof enzyme
1 Substrates enter active site in aspecific orientation.
2 Substrates and active sitechange shape, promotingreaction between substrates.
enzyme
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Structural Proteins are Functional
Proteomics work flow and technologies
2-dimensional gel based technologiesMass spectrometry for protein identificationMass spectrometry for protein quantification
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2-dimensional gel based technologies
Human Proteome
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Theoretical pI and M r map of yeast cell proteins
1
10
100
1000
2 4 6 8 10 12 14
Theoretical pI
M r / k D a
From: Wildgruber et al. Electrophoresis. 21 (2000) 2610-2616 .
2-D Gel Electrophoresis
1 st dimension: isoelectric focusing (IEF)- separate proteins based on their isoelectric point (pI) byusing immobilized pH gradient (IPG) gel strips.
2nd dimension: SDS PAGE- separate proteins based on their molecular mass
A powerful technique for protein separation
IEFSDS
pI 3 10
25
37
50
75
100
150200
MW kDa
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2-D image analysis
Control Treated
Difference in Gel Electrophoresis (DIGE)
ProteinExtract 1
Label withCy2
ProteinExtract 2
Label withCy3
ProteinExtract 3
Label withCy5
Mix labeled extracts
2D gel separation
Cy2 Cy3 Cy5
Analysis of Differences
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Protein Expression -Bioinformatics
Expression Analysis
B
Excise spot; elute; digestExtract peptides;MS analyzeProtein identification
A
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Ions formation
ion source mass analyzer ion detector
Data System
m/z
Separation Detection
Inlet
Sample Introduction
mass spectrum
Overview of Mass Spectrometry
Amino Acid Residue Mass: crucial for mass spectrometry-based protein identification and peptide sequencing
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tryptic digestioncleaves protein atR and K residues
Peptide Mass Fingerprinting(PMF) spectrum
Sample Preparation
Gel Electrophoresis
Cut spots
(peptides are fragmentedin mass spectrometer)
MS/MS analysis
MS/MS Peptide Sequencing Spectra
MS Analysis
Peptide Mass Mapping
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1901.872
1875.97
1838.932
1707.789
1499.816
1498.81
1493.742
1490.765
1489.751
1488.756
1475.757
1418.816
1417.729
1404.783
1401.795
1353.7
1350.694
1320.608
1308.659
1236.56
1235.564
1234.663
1180.588
1179.593
1060.516
1058.51
1045.558
1000.446
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How does the search engine works?
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How does the search engine works?
842.509
870.461
882.43
886.448
886.959
898.453
905.657
927.475
937.456
965.484
974.44
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
2225.12
2239.14
2344.09
2359.18
2458.18
2566.18
2807.31
2985.48
Bovine Serum Albumin peptide2-oxoglutarate dehydrogenase peptide
An example of a spectrum in which not all of the major peaks matched theprimary identification. A second component search reveals the identity of the other protein.
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Tandem Mass Spectrometry
Tandem Mass Spectrum: An Example
Secondary Fragmentation
Ionized parent peptide
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Tandem Mass Spectrometer
massanalyzer
fragment
precursor ions fragment ions
MPSER
SG
+
PAK +
+
P + AK PAK +
PAK + PA + K
AK +P
K +PA
P +K +
PA+
AK +
PAK +
PAK +
d e n o v o s e q u e n c i n g
massanalyzer
ionsdetector
N- and C-terminal Peptides
N - t e r
m i n a
l p e p t i
d e s
C - t e
r m i n a
l p e p t i d
e s
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How Does a Peptide Fragment?
m(y 1)=19+m(A 4)m(y 2)=19+m(A 4)+m(A 3)m(y 3)=19+m(A 4)+m(A 3)+m(A 2)
m(b 1)=1+m(A 1)m(b 2)=1+m(A 1)+m(A 2)m(b 3)=1+m(A 1)+m(A 2)+m(A 3)
MS/MS Peptide Sequencing
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Peptide sequencing by MS/MS
Peptide: [Glu1
]-Fibrinopeptide B (EGVNDNEEGFFSAR MW 1569.7844)
MS/MS database search
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A-D-L-V-I-P-N-D-F-K
1320.584225.102
233.189
255.192261.144382.237399.228
434.255462.279
632.300
649.399662.368
688.550809.452
843.397
860.417
1320.589178.117
195.104
223.147251.210258.162436.213
450.349484.248
485.234
545.357920.441
953.4501057.401
1066.465
1075.395
1320.560200.382
219.491
251.040282.611290.432490.740
506.642544.779
545.888
613.5261035.496
1072.6311189.576
1199.773
1320.583169.745185.934
212.659239.403246.028415.711
429.182461.488
462.428
519.725877.180
908.6381007.703
1016.341
1320.586164.046179.690
205.518231.364237.767401.752
414.771445.992
446.900
502.273847.726
878.128973.867
982.215
1320.584225.102233.189
255.192261.144382.237399.228
434.255462.279
632.300
649.399662.368
688.550809.452
843.397
860.417
1320.589178.117195.104
223.147251.210258.162436.213
450.349484.248
485.234
545.357920.441
953.4501057.401
1066.465
1075.395
1320.560200.382219.491
251.040282.611290.432490.740
506.642544.779
545.888
613.5261035.496
1072.6311189.576
1199.773
1320.583169.745185.934
212.659239.403246.028415.711
429.182461.488
462.428
519.725877.180
908.6381007.703
1016.341
1320.586164.046179.690
205.518231.364237.767401.752
414.771445.992
446.900
502.273847.726
878.128973.867
982.215
1320.585178.128
195.166
258.121312.219
329.204436.219
450.330466.292
545.365
694.343
920.455
1057.4271066.477
1075.6491283.684
vs.
Database Search of MS/MS Data
Databases:
Protein databases:
Swiss-Prot: Curated protein databasehigh quality, low coverage, modification information
TrEMBL: translated EMBL cDNA databasemedium quality, medium coverage
Nucleotide database:
Expressed sequence tag (dbEST)low quality, high coverage
Genome sequencehigh quality, everything included, needs prediction
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Protein Databases
Compiled from a variety of resources:Translations from annotated codingregions in GenBank and RefSeq.SwissProtPIR (Protein Information Resource)PDB (Protein DataBank 3D structures)
6,417,545Total
67,416PDB
234,946PIR12,079PRF
185,800Swiss Prot
4,168Third Party Annotation
1,616,076RefSeq4,297,060GenPept
IPI database
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Application 1: Protein Expression Level Changes
Use Proteomics to Investigate Molecular Mechanismsof Aging and Gender differences in the Heart
Yan et al. J Mol Cell Cardiol. 2004 Nov;37(5):921-9.
Objective
To reveal aging and gender-associated alterations inprotein expression and function that could explain whyfemale life expectancy is generally greater than malesand particularly why their cardiovascular risk is less.
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pH 5 8
25
37
50
75
100150200
kDa
3-Oxoacid CoA transferase
Acyl-CoA dehydrogenase
-enolase
Triosephosphate isomerase
Oxoglutarate dehydrogenase
1
2
3
4
5 1
2
3
4
5
YM OM YF OF
Spot No. Proteins1 3-Oxoacid CoA transferase2 Acyl-CoA dehydrogenase3 -enolase4 Triosephosphate isomerase5 Oxoglutarate dehydrogenase
Alterations in LV total protein expression in aging monkeys
a
b
c
a
b
c
1 1 1 1
2 2 2 2
3 3 3 3
4 4 4 4
YM OM YF OF
Spot No. Proteins1 ATP-spec if ic succ inyl -CoA synthetase subunit2 Pyruvate dehydrogense E1 subunit3 ATP synthase subunit4 Acyl-CoA dehydrogenase
25
37
50
75
100
150200
kDa
pH 5 8
Alterations in mitochondrial protein expression in aging monkeys
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Table 1. Alterations in Protein Expression Related to Metabolic Pathways
Proteins AccessionNo.MetabolicPathway OM
vs YM * OF vs YF * OM vs OF * DetectionMethods
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)
Pyruvate kinase P14618P14786 glycolysis WBPyruvate dehydrogense E1 subunit P11177 Glucose oxidation 2DGE(M)
Oxoglutarate dehydrogenase Q02218 TCA cycle 2DGE(T)ATP Specific succinyl-Co Asynthetase subunit Q9P2RT TCA cycle 2DGE(M)
ATP synthase 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
Decreased expression of metabolic enzymes related to energyproduction in aging male monkeys
Proteomic Alterations of Cardiac Troponin T, a Novel Mechanismfor the Transition from Hypertrophy to Heart Failure
Application 2: Protein Post-translational Modification (proteolysis)
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Background
Clinically, the decompensated stage of heart failure (HF) isusually preceded by chronic, compensatory cardiachypertrophy.
The molecular mechanisms that precipitate the transitionfrom stable hypertrophy to failure remain largely unknown.
A Proteomic Approach to Determine the Alterations in ProteinExpression in Canine Model
Protein ID
LV tissues: Normal, LVH, LVH/HF
Protein extracts
2D gel electrophoresis (1 st : IEF 2 nd : SDS-PAGE)
Gel comparison by computer image analysis
Selected protein spots
Tryptic peptides
Peptide masses
Sequence database search
Homogenization
Protein visualization by Sypro Ruby
Spots excisionIn-gel digestion with trypsin
MALDI-TOF MSPeptide sequence
Q-TOF MS/MSMALDI-TOF/TOF MS/MS
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75
50
37
25
MW (kDa)
CB
37
25
B C
LVH LVH/HFNormal
pH 5 8 5 8 5 8
pH 5 5.5 5 5.5 5 5.5
89 10
7
12345
6
A
7
12345
6
Alterations of Cardiac Troponin T (cTnT) Expressionin Hypertrophy (LVH) and Heart Failure (LVH/HF)
Identification of cTnT
MALDI mass spectrum of in-gel trypsin digest of spot 5 shown on last slide
796.0 1191.8 1587.6 1983.4 2379.2 2775.0
Mass (m/z)
0
5.8E+4
0
10
20
30
40
50
60
70
80
90
100
% I
n t e n s
i t y
Voyager Spec #1 MC[BP = 1943.0, 58237]
1943.0047
1535.8264
906.49302466.1986
1945.98421629.7696
1683.8241
1538.8141 2416.18421622.81521155.6182 2469.20211408.8022
842.4784 1812.89271632.7437
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Data interpretation and advanced application
Phosphorylation site identificationProtein complex identificationProtein isoform problemLimitation of current technologiesEstimation of sample requirement
MS-based peptide quantification
Phosphorylation 80Acetylation 42Methylation 14Hydroxy amino acids 16Acylation
Myristic acid 228Palmitic acid 256
PrenylationFarnesol 204Geranylgeranol 272
Nitrosylation 39 or 45Oxidation 16Other oxidation -32 loss of SH
Dityrosine formationIsoaspartate 1 (+shift of peptide bond)
Glycation variableGlycoxidation variableLipid peroxide adduction variable
Posttranslational Modifications (PTM)
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Isolating Modified Peptides by Affinity Chromatography Prior to Tandem Mass Spectrometric Characterization
Selective enrichment of phosphopeptides with Immobilized Metal AffinityChromatography (IMAC)
Proteintrypsin
Tryptic peptidesIMAC
Purified phosphopeptides
MS/MSsequencing
Phosphorylation sites
HPLC MALDI-TOF MS
before IMAC before IMAC
after IMACafter IMAC
(A)
(B)
(C)
1100 1490 1880 2270 2660 3050Mass(m/z)
3336.9
0
10
20
30
40
50
60
70
80
90
100
% I
n t e n s i
t y
4700Reflector Spec#1[BP = 1591.9,3337]
1100 1490 1880 2270 2660 3050Mass(m/z)
8366.3
0
10
20
30
40
50
60
70
80
90
100
% I
n t e n s
i t y
4700Reflector Spec#1[BP = 2061.8,8366]
276 655 1034 1413 1792 2171Mass(m/z)
921.4
0
10
20
30
40
50
60
70
80
90
100
% I
n t e n s
i t y
4700MS/MS Precursor2062 Spec#1=>NR(30.00)=>NR(50.00)[BP = 1965.3,921]
1 1 3 7
. 5 6
1 2 6 7
. 7 1
1 3 8 4
. 7 3
1 5 9 1
. 9 4
1 8 8 1
. 0 8
2 0 6 1
. 8 4
2 1 3 2
. 0 7
2 1 8 6
. 1 7
2 3 3 0
. 5 7
2 4 2 4
. 1 6
2 6 2 6
. 4 9
2 9 0 9
. 6 1
2 0 6 1
. 8 4
1 9 5 8
. 1 8
2 0 8 3 . 8 1
2
3 3 0
. 5 7
2 4 2 4
. 1 6
2 9 0 9
. 6 1
1 2 1 3
. 0 7
1 6 6 0
. 7 9
503.37
632.43 747.47876.53
977.59
1105.68
1233.74
1361.841490.98
1619.97
1786.93
1964.39
y4
y5 y6
y7
y8
y8
y10y11
y12y13
y142061.8
KDQL E D E T Q Q Q E E pS QF
Before IMAC
After IMAC
H3
P O
4
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Study of Protein-Protein Interactions using MassSpectrometry-Based Methods
Isolation of protein complexes by affinity approaches
1D/2D GE or HPLC
Trypsin digestion
Protein identification by mass spectrometry
Affinity purification + Mass spectrometry identification
Affinity approaches for the retrieval of proteincomplexes
ImmunoprecipitationEpitope-tagging (Myc, HA, Flag, KT3)GST-pulldown
Tandem affinity purification (TAP)
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2D gel of spliceosome-associated proteins
SPF45
GFP-SPF 45
Localization of SPF 45 in nucleus
GST
GST-S14 GST beads
Meaning of identifying a protein
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Protein isoform problem
Meaning of quantifying a protein
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Limitation of Current Technology
Protein detection on gel
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How Much Sample is Enough?
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