2009 introduction of proteomics 20090502

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Copyright 2008 ITRI 1

Proteomics and biomedicalProteomics and biomedicalapplicationapplication

Introduction of Proteomics

[email protected] 02, 2009




Outline

The characters of proteinsWhy to study proteomeProteomics

Introduction to proteomics

The major techniques in current proteomicsPost- translational ModificationProtein- protein interactionQuantitative Proteomcis by Massproteomics database

Major Directions in Coming Proteomics




Synthesis of DNA and protein

DNA

Proteins

Sugar Chain




Mechanisms by which a single gene can give

rise to multiple gene products




Three developments formed thefoundation of the new biology

The growth of gene, expressed sequence tag (EST), andprotein-sequence databases during the 1990s.

The introduction of user-friendly, browser-basedbioinformatics tools .

The development of oligonucleotide microarray .




Proteins are the molecule tools formost cellular functions

TYPE FUNCTION EXAMPLEStructural proteins Support Collagen, Elastin,

KeratinStorage proteins Storage of amino acid Ovalbumin, CaseinTransport proteins Transport of other

substrateHemoglobin

Hormonal proteins Coordination of andorganism’s activities

Insulin

Receptors proteins Response of cell tochemical stimuli

Receptor in nervetransmit route

Contractile proteins Movement Actin, Myosin

Defensive proteins Protecton against disease AntibodysEnzymatic proteins Selective acceleraton of

chemical reactionsTrypsin, ATPase,GAPDH




Why the transcriptomic analysesmay not have revealed all proteins ?

lack of correlation between transcript anddisease- associated protein levelstranslocation of a protein in the disease staterather than simply differential levels of thetranscriptnovel/uncharacterized genes that are not highlyrepresented within the "closed system" of acDNA array




DNA

mRNA

Proteins

Metabolyte

Genome“Genomics”

Proteome“Proteomics”

Omics

Transcriptome“Transcriptomics”

Metabolome“Metabolomics”




-omicsGenomics ( ) -study of genome, quantitative study of genes,regulatory and noncoding sequencesTranscriptomics ( ) -study of RNA and gene expressionProteomics ( ) -study of proteome, study of proteinexpressionMetabonomics/Metabolomics ( ) -study of metabolites andmetabolic networkPharmacogenomics -quantitative study of how genetics affectshost’s response to drugsPhysiomics -physiological dynamics and functions of wholeorganismsBioinformatics ( )




Genomics, Proteomics,& Systems Biology

Systems Biology

Proteomics

Genomics

1990 2000 2010 2020

Dr. David Wishart




What’s Proteomics?




What’s “proteomics” ?

"The analysis of the entire PROTE in complement

expressed by a gen OME , or by a cell or tissue type.“

Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium .

Electrophoresis 16 (1995) 1090-1094

Two MOST related technologies:1. 2-D electrophoresis : separation of complex

protein mixtures2. Mass spectrometry : Identification and structure

analysis




Proteomics Approach




Generalized proteomics scheme

Yarmush & Jayaraman, 2002




Definitions of ProteomicsBe defined as the large- scalecharacterization of the entire proteincomplement of a cell line, tissue, or organism.Goal:- To obtain a more global and integrated viewof biology by studying all the proteins of acell rather than each one individually.




Defining proteomics

The classical definitionTwo-dimensional gels of cell lysate and annotationTwo-dimensional gels to visualize differential protein expressionProtein Identification

In the post-genomics eraPost-translational modificationsDetermining FunctionStructure proteomicsMolecular Medicine

Differential display by two-dimensional gelsProtein-Protein Interactions




Theoretical pI and M r map of yeast cell proteins(total 6360 proteins)

(calculated from MIPS data)Theoretical pI

M r /

k D a




Why is Proteomics necessary?

Having complete sequences of genome is not sufficient toelucidate biological function .A cell is normally dependent upon multitude of metabolicand regulatory pathways for its survivalModifications of proteins can be determined only byproteomic methodologiesIt is necessary to determine the protein expression levelThe localization of gene products can be determinedexperimentallyProtein- protein interactions

Proteins are direct drug targets .




The Major Techniques inCurrent Proteomics

Two- dimensional electrophoresisIEF strip separationSDS- PAGE gel separation

Mass SpectrometryProtein sequencing

Peptide mappingPost- translational Modification

OthersICAT Yeast two hybrid assayProtein chips




Ionization State as a Function of pH




separates proteins by charge (separates proteins by charge ( isoelectric pointsisoelectric points ) by) byelectrophoresis through a pH gradient in a polyacrylamide stripelectrophoresis through a pH gradient in a polyacrylamide strip

+-On electrophoresis, each protein migrates throughOn electrophoresis, each protein migrates through

pH gradient to point where its net charge is 0pH gradient to point where its net charge is 0

Isoelectric focusing Isoelectric focusing

First DimensionFirst Dimension




separates proteinsseparates proteins orthogonallyorthogonally by theirby their molecular massesmolecular masses in anin anSDSSDS --polyacrylamide gelpolyacrylamide gel

-

+

Second DimensionSecond Dimension




First dimension: IEF (based on isoelectric point)

S D

S -P A GE

( b a s e d onm

ol

e c ul a r

w e i gh t )

+ -

acidic basic

HighMW

LowMW

Sample

Two-dimensional GelElectrophoresis




Silver staining Coomassie blue staining Sypro Ruby staining

Staining of Polyacrylamide Gels




Image Analysis




Hydrophilic proteins (pH 3~10)




Spot 900- 3D Viewer

900

Control

LPS-treated




In- gel digestion- trypsin, chymotrypsin, Glu C, Lys C, Asp N, …




1. Cut protein spot 2. Protein digestion

3. Peptide purification4. Spot onto MALDI chip

5. MALDI-TOF analysis 6. Peptide fragment fingerprint

Protease

Protein Identification by MALDI-TOF




*

*

**

**

*

Trypsin

Peptide mass fingerprinting (PMF) or peptide mappingPeptide mass fingerprinting (PMF) or peptide mapping

Mass SpectrometricMass Spectrometric Identification ofIdentification ofProteinsProteins -- MappingMapping




MS analysis




Typical mass spectrometry schemepeptide mass fingerprint & tandem mass spectrometry

Yarmush & Jayaraman,2002




IonizationIonizationSampleSampleinputinput AnalyzerAnalyzer DetectorDetector

HowHow DDoes aoes a MMassassSSpectrometerpectrometer WWork?ork?




Sample Input:Sample Input:Gas Chromatography (GC), Liquid ChromatographyGas Chromatography (GC), Liquid Chromatography(LC), Capillary Electrophoresis (CE), Solid crystal(LC), Capillary Electrophoresis (CE), Solid crystaletc.etc.

Ionization:Ionization:ElectrosprayElectrospray , Matrix, Matrix --assisted Laserassisted Laser DesorptionDesorption //IonizationIonization ((MALDI) etcMALDI) etc

Analysis:Analysis:QQuadrupoleuadrupole ,, T T ime ofime of Flight (TOF)Flight (TOF) , ion trap etc., ion trap etc.

Detection:Detection:Electron multiplier, Scintillation counterElectron multiplier, Scintillation counter

The basic components of a massspectrometry system




MALDI Mass Spectrometer




Matrix Assisted Laser Desorption(MALDI)




Time of flight Mass Spectrometry




SELDI (Surface-Enhanced Laser Desorption/Ionization)

Crude sample is placed (and

processed) on a ProteinChip Array

Proteins bind to chemical or biological“docking sites” on the ProteinChipsurface.

WASHNon-binding proteins, salts,and other contaminants arewashed away, eliminatingsample “noise”

D e t e c t o rLa ser

TOF-MS

2000

2000 4000 6000 8000

4000 6000 8000 Gel View

02.5

57.5 Spectra View

80002000 40006000

2

4

6

2185.8+H

2369.9+H2528.2+H

2781.9+H3172.3+H

3915.2+H4000.2+H

4345.6+H

4618.2+H

4730.4+H

5045.2+H

7977.1+H

Map View

Retained proteins are “eluted” from the ProteinChip Array by Laser Desorption and IonizationIonized proteins are detected and their mass accurately determined by Time-of-Flight Mass Spectrometry




52 2004-06-25

SELDI

( 92%, 97%)SELDI--

( 80%, 86%)SELDI( 40%)( )

( 85%, 82%)SELDI( <50%): CA199

( 83%, 92%)SELDI( 47%): CEA

( 99%, 99%)SELDI( 34%): CA125

( 93%, 91%)SELDI( 23%): CA153

( 83%, 97%)SELDI( 90% 25%): PSA

( 91%, 94%)SELDI( <50%): CEA

( 82%, 95%)SELDI( <50%): NSE

( 91%, 89%)SELDI( 42%): AFP




Microarrays for Genomics and Proteomics

DNA microarray are usedfor genetic analysis aswell as expressionanalysis at the mRNA

level.Protein microarrays areused for expressionanalysis at the proteinlevel and in the expansivefield of interactionanalysis.




‘proteome chip’ composed of 6,566 protein samples

representing 5,800 unique proteins, which are spotted induplicate on a single nickelcoated glass microscope slide39. Theimmobilized GST fusion proteins were detected using a labeledantibody against GST.

(Nat Genet, 2002;32 S2: 526-32 )

Proteome chip




Protein Microarray in MedicalResearch

Accelerate new drug developmentThe reduction of sample volume

the analysis of multiple tumor markers from a minimumamount of biopsy material.

New possibilities for patient monitoringduring disease treatment and therapy willbe developed based on this emergingtechnology.




Clinical and Biomedical Applications ofProteomics

An approach complementary to genomics is required inclinical situationsThe clinical applications of 2- D PAGE & MS

Identifying the origin of body fluid samples or theorigin of a tissue biopsy .

Analyzing protein phenotypes and protein post-translational modifications in fluid, cells, or tissues.Examining the clonality of immunoglobulins anddetecting clones which are not seen with conventionaltechniques.Monitoring disease processes and protein expression.Discovering new disease markers and/or patterns in

body fluids, cells, or tissues.




Clinical applications of2- D electrophoresis

Body fluidsBlood cellPlasma and serumUrineCerebrospinal fluidAmniotic fluidSynovial fluidSalivaSweatTearsSemen

Solid tissueHeartBrainThyroidMuscle

Malignant diseases

Tissue cultureMalignant cellsBacterial proteins

Young & Tracy Journal of Chromatography A, 698 (1995) 163-179




Examples of posttranslational modifications in proteinsCarbohydrates

N-linked oligosaccharidesO-linked oligosaccharidesGlycophosphoinositol-lipid anchorsC-Mannosylation

LipidsAcylationPalmitylationMyristylationFarnesylationPrenylation

GeranylgeranylationAmino acid modification

Amidation–deamidationCysteinylationFormylationCitrullinationIsoaspartylationDetyrosinationThioether additionGamma-carboxylationDeimidation

Cross-linkingDisulfide formationThiolester formationTransglutamylation

Small adductsPhosphorylationSulfationNitrationAcetylationFormylationMethylation

OxidationHydroxylationIodinationBiotinylation

MiscellaneousComplex formation (noncovalent or covalent)PolymerizationConformational changesFolding into different formsProteolytic cleavagesUbiquitinylation




The post- translational modification

in proteome analysis2D PAGEIncorporation of a radiolabel

HPLCEdman degradationMass spectrometer




Serial dichromatic detection of glycosylated and un-glycosylated proteins in the same polyacrylamide gel

Pro-Q Emerald 488 glycoprotein stain kit SYPRO Ruby protein gel stain

Glycoproteins Total proteins




Multiplexed analysis of phosphorylation, glycosylation, andprotein expression levels in the same 2-D gel

Phosphoproteins(Pro-Q Diamond)

Total proteins(SYPRO Ruby)

Glycoproteins(Pro-Q Emerald)

Proteomics.

2003;3(7):1196



Copyright 2008 ITRI 55NATURE, VOL 405, 15 JUNE 2000

A strategy for mass spectrometric identification ofproteins and post-translational modifications



Copyright 2008 ITRI 56Pandey A & Mann M, 2000

A strategy for mass spectrometric identification ofproteins and post-translational modifications




Chemical modification

Covalent linkage of chemical group to aminoacid in proteinSeveral types:

Acetylation (common on first amino acid)Phosphorylation (often involved in regulation)Lipidation (attachment to membrane)Glycosylation (common outside cell)




Mass ChangeModification

-2.0 Disulfide bond formation+14.0 Methylation+16.0 Hydroxylation

+28.0 Formylation+30.0 Nitrosylation+42.0 Acetylation+80.0 Sulfation+80.1 Phosphorylation+180 Mono-glycosylation+204.4 Farnesylation+210.4 Myristoylation

Mass Changes from Post-translational Modification




Protein-protein interactions

Protein- protein interactions areintrinsic to every cellular process.Form the basis of phenomena

DNA replication and transcription

MetabolismSignal transductionCell cycle controlSecretion




The Study of Protein-protein Interactionby Mass Spectrometry

bai t

S14

?

???

* ***

SDS- PAGE

MASS




Yeast Two- hybrid System




Interaction maps - Grid




Quantitative Proteomcis

SILAC-cell culture, in vivo labelingStable Isotope Labeling by Amino Acids in Cell Culture.

ICAT-protein, pre-digestion in vitro labelingIsotope-coded affinity tagging

ITRAQ-peptide, post-digestion in vitro labelingis an amine-specific peptide-based labeling technology for simultaneousidentification and quantitation of biological samples.




Quantitative proteomics procedures using Mass

Curr Opin Chem Biol. 2003;7, 70




Isotopically-coded affinitytagging (ICAT)




amine-specific isobaric tagging (iTRAQ)




SILAC TM




ExPASy Proteomics tools(http://www.expasy.ch/tools/)

Protein identification and characterizationIdentification and characterization with peptide mass fingerprintingdata (9)Identification and characterization with MS/MS data (7)Identification with isoelectric point, molecular weight and/or aminoacid composition (4)Other prediction or characterization tools (6)

Other proteomics toolsOther tools for MS data (vizualisation, quantitation, analysis, etc.) (3)Other tools for 2-DE data (image analysis, data publishing, etc.) (2)

DNA -> Protein (8)Similarity searches (15)Pattern and profile searches (16)Post-translational modification prediction (33)

Topology prediction (13)




ExPASy Proteomics tools(http://www.expasy.ch/tools/)

Primary structure analysis (28)Secondary structure prediction (16)Tertiary structure

Tertiary structure analysis (5)Tertiary structure prediction (16)Assessing tertiary structure prediction (6)Molecular modeling and visualization tools (10)Prediction of disordered regions (3)

Sequence alignmentBinary (3)Multiple (12)Alignment analysis (11)

Phylogenetic analysis (11)Biological text analysis (5)




Major Directions in Coming Proteomics

Chemical proteomics (screens for activity and binding)Structural proteomics (target validation anddevelopment)Interaction proteomics (identification of new proteintargets)Protein structure prediction and modeling

Assignment of protein structure to genomesClassifications of protein structuresDrug discovery and developmentBioinformatics (annotation of the proteome)



Types of Proteomics and TheirApplications to Biology

2009 introduction of proteomics 20090502

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