methods in cell biology proteomics _ hong ki et al lecture

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

[email protected]

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?

methods in cell biology proteomics _ hong ki et al lecture

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