Protein-protein interactionsProtein-protein interactions“The Interactome”“The Interactome”

Yeast two-hybrid analysisYeast two-hybrid analysis Protein chipsProtein chips Biochemical purification/Mass Biochemical purification/Mass

spectrometryspectrometry

Yeast two-hybrid methodYeast two-hybrid method Goal: Determine how proteins interact with each otherGoal: Determine how proteins interact with each other MethodMethod

Use yeast transcription factorsUse yeast transcription factors Gene expression requires the following:Gene expression requires the following:

A DNA-binding domainA DNA-binding domain An activation domainAn activation domain A basic transcription apparatusA basic transcription apparatus

Attach proteinAttach protein11 to DNA-binding domain (bait) to DNA-binding domain (bait) Attach proteinAttach protein2 2 to activation domain (prey)to activation domain (prey) Reporter gene expressed only if proteinReporter gene expressed only if protein11 and protein and protein22

interact with each otherinteract with each other

A schematic of the yeast two-A schematic of the yeast two-hybrid methodhybrid method

m

n

Results from a yeast two-hybrid Results from a yeast two-hybrid experimentexperiment

Goal: To characterize protein–protein Goal: To characterize protein–protein interactions among 6,144 yeast ORFsinteractions among 6,144 yeast ORFs 5,345 were successfully cloned into 5,345 were successfully cloned into

yeast as both bait and preyyeast as both bait and prey Identity of ORFs determined by DNA Identity of ORFs determined by DNA

sequencing in hybrid yeastsequencing in hybrid yeast 692 protein–protein interaction pairs692 protein–protein interaction pairs Interactions involved 817 ORFsInteractions involved 817 ORFs

Yeast two-hybrid results for Yeast two-hybrid results for flies & wormsflies & worms

Worms:Worms: Created >3000 bait constructsCreated >3000 bait constructs Tested against two AD librariesTested against two AD libraries Mapped 4000 interactionsMapped 4000 interactions

Flies:Flies: Screened 10,000 predicted transcriptsScreened 10,000 predicted transcripts Found 20,000 interactionsFound 20,000 interactions

Statistically assigned 4800 as “high quality” Statistically assigned 4800 as “high quality” interactionsinteractions

Caveats associated with the Caveats associated with the yeast two-hybrid methodyeast two-hybrid method

There is evidence that other methods There is evidence that other methods may be more sensitivemay be more sensitive

Some inaccuracy reported when Some inaccuracy reported when compared against known protein–compared against known protein–protein interactionsprotein interactions False positivesFalse positives False negativesFalse negatives

Protein chipsProtein chips Thousands of proteins Thousands of proteins

analyzed analyzed simultaneouslysimultaneously

Wide variety of assaysWide variety of assays Antibody–antigenAntibody–antigen Enzyme–substrateEnzyme–substrate Protein–small moleculeProtein–small molecule Protein–nucleic acidProtein–nucleic acid Protein–proteinProtein–protein Protein–lipidProtein–lipid Yeast proteins detected

using antibodies

Fabricating protein chipsFabricating protein chips

Protein substratesProtein substrates Polyacrylamide or Polyacrylamide or

agarose gelsagarose gels GlassGlass NanowellsNanowells

Proteins deposited Proteins deposited on chip surface by on chip surface by robotsrobots

Protein attachment Protein attachment strategiesstrategies

DiffusionDiffusion Protein suspended in Protein suspended in

random orientation, but random orientation, but presumably activepresumably active

Adsorption/AbsorptionAdsorption/Absorption Some proteins inactiveSome proteins inactive

Covalent attachmentCovalent attachment Some proteins inactiveSome proteins inactive

AffinityAffinity Orientation of protein Orientation of protein

precisely controlledprecisely controlled

Diffusion

Adsorption/Absorption

Covalent

Affinity

Classes of capture Classes of capture moleculesmolecules

Different capture Different capture molecules must be molecules must be used to study different used to study different interactionsinteractions

ExamplesExamples Antibodies (or antigens) Antibodies (or antigens)

for detectionfor detection Proteins for protein-Proteins for protein-

protein interactionprotein interaction Enzyme-substrate for Enzyme-substrate for

biochemical functionbiochemical function Receptor–ligand

Antigen–antibody

Protein–protein

Aptamers

Enzyme–substrate

Reading out resultsReading out results FluorescenceFluorescence

Most common methodMost common method Fluorescent probe or tag Fluorescent probe or tag Can be read out using standard nucleic acid microarray Can be read out using standard nucleic acid microarray

technologytechnology Surface-enhanced laser desorption/ionization (SELDI)Surface-enhanced laser desorption/ionization (SELDI)

Laser ionizes proteins captured by chipLaser ionizes proteins captured by chip Mass spectrometer analyzes peptide fragmentsMass spectrometer analyzes peptide fragments

Atomic-force microscopyAtomic-force microscopy Detects changes in chip surface due to captured proteinsDetects changes in chip surface due to captured proteins

Difficulties in designing protein Difficulties in designing protein chipschips

Unique process is necessary for Unique process is necessary for constructing each probe elementconstructing each probe element

Challenging to produce and purify Challenging to produce and purify each protein on chipeach protein on chip

Proteins can be hydrophobic or Proteins can be hydrophobic or hydrophilichydrophilic Difficult to design a chip that can detect Difficult to design a chip that can detect

bothboth

Purification of interacting Purification of interacting proteinsproteins

ImmunoprecipitationImmunoprecipitation Impractical on large scale (identification Impractical on large scale (identification

of unknowns)of unknowns) Affinity purificationAffinity purification

Biochemically practical, but too dirtyBiochemically practical, but too dirty Tandem affinity purificationTandem affinity purification

Sufficient yield & purity for identification Sufficient yield & purity for identification of unknown proteinsof unknown proteins

TAP TAP Purification Purification

StrategyStrategy

Identification of Interacting Identification of Interacting ProteinsProteins

ProteolyticDigestion(Trypsin)

MassSpectrometric

Analysis

Identifying proteins with mass Identifying proteins with mass spectrometry spectrometry

Preparation of protein samplePreparation of protein sample Extraction from a gelExtraction from a gel Digestion by proteases — e.g., trypsinDigestion by proteases — e.g., trypsin

Mass spectrometer measures mass-charge ratio Mass spectrometer measures mass-charge ratio of peptide fragmentsof peptide fragments

Identified peptides are compared with databaseIdentified peptides are compared with database Software used to generate theoretical peptide mass Software used to generate theoretical peptide mass

fingerprint (PMF) for all proteins in databasefingerprint (PMF) for all proteins in database Match of experimental readout to database PMF Match of experimental readout to database PMF

allows researchers to identify the proteinallows researchers to identify the protein

Mass Mass spectrometryspectrometry

Measures mass-to-Measures mass-to-charge ratiocharge ratio

Components of Components of mass spectrometermass spectrometer Ion sourceIon source Mass analyzerMass analyzer Ion detectorIon detector Data acquisition Data acquisition

unitunit

A mass spectrometer

Principle of mass Principle of mass spectrometryspectrometry

Ion sources used for Ion sources used for proteomicsproteomics

Proteomics requires Proteomics requires specialized ion sourcesspecialized ion sources

Electrospray Ionization Electrospray Ionization (ESI)(ESI) With capillary With capillary

electrophoresis and electrophoresis and liquid chromatographyliquid chromatography

Matrix-assisted laser Matrix-assisted laser desorption/ionization desorption/ionization (MALDI)(MALDI) Extracts ions from Extracts ions from

sample surfacesample surface

ESI

MALDI

Mass analyzers used for Mass analyzers used for proteomicsproteomics

Ion trapIon trap Captures ions on the basis Captures ions on the basis

of mass-to-charge ratioof mass-to-charge ratio Often used with ESIOften used with ESI

Time of flight (TOF)Time of flight (TOF) Time for accelerated ion Time for accelerated ion

to reach detector to reach detector indicates mass-to-charge indicates mass-to-charge ratioratio

Frequently used with Frequently used with MALDIMALDI

Also other possibilitiesAlso other possibilities

Ion Trap

Time of Flight

Detector

A mass spectrumA mass spectrum

Identifying proteins with mass Identifying proteins with mass spectrometry spectrometry

Preparation of protein samplePreparation of protein sample Extraction from a gelExtraction from a gel Digestion by proteases — e.g., trypsinDigestion by proteases — e.g., trypsin

Mass spectrometer measures mass-charge ratio Mass spectrometer measures mass-charge ratio of peptide fragmentsof peptide fragments

Identified peptides are compared with databaseIdentified peptides are compared with database Software used to generate theoretical peptide mass Software used to generate theoretical peptide mass

fingerprint (PMF) for all proteins in databasefingerprint (PMF) for all proteins in database Match of experimental readout to database PMF Match of experimental readout to database PMF

allows researchers to identify the proteinallows researchers to identify the protein

Limitations of mass Limitations of mass spectrometryspectrometry

Not very good at identifying minute Not very good at identifying minute quantities of proteinquantities of protein

Trouble dealing with phosphorylated Trouble dealing with phosphorylated proteinsproteins

Doesn’t provide concentrations of Doesn’t provide concentrations of proteinsproteins

Improved software eliminating human Improved software eliminating human analysis is necessary for high-throughput analysis is necessary for high-throughput projectsprojects

Protein ComplementationProtein Complementation

Enzymatic complementationEnzymatic complementation -galactosidase reconstitution-galactosidase reconstitution

Fluorescence complementationFluorescence complementation GFP or YFP reconstitutionGFP or YFP reconstitution FRET (fluorescence resonance energy FRET (fluorescence resonance energy

transfer)transfer)

Enzymatic Enzymatic ComplementationComplementation

Blue=DAPI

Red=BGAL

Blue=DAPI

Green=BGAL

Red=Actin

Bimolecular Fluorescence Bimolecular Fluorescence ComplementationComplementation

FRET (fluorescence resonance FRET (fluorescence resonance energy transfer)energy transfer)

Pregenomics biochemical Pregenomics biochemical assaysassays

Methods used to find genes responsible Methods used to find genes responsible for specific biochemical activity before the for specific biochemical activity before the inception of genomicsinception of genomics Laboriously purify responsible proteinLaboriously purify responsible protein

Often expensive and time consumingOften expensive and time consuming Expression cloningExpression cloning

Introduce cDNA pools into cellsIntroduce cDNA pools into cells Look for biochemical activity in those cellsLook for biochemical activity in those cells Caveat: Often difficult to detect biochemical Caveat: Often difficult to detect biochemical

activity in cell’s biochemical “background”activity in cell’s biochemical “background”

Biochemical genomicsBiochemical genomics( “Enzomics”? )( “Enzomics”? )

Genome of an organism is already Genome of an organism is already knownknown

ApproachApproach Construct plasmids for all ORFsConstruct plasmids for all ORFs

Attach ORFs to sequence that will facilitate Attach ORFs to sequence that will facilitate purificationpurification

Transform cellsTransform cells Isolate ORF productsIsolate ORF products Test for biochemical activityTest for biochemical activity

Biochemical genomics in Biochemical genomics in yeastyeast

6,144 ORF yeast strains made6,144 ORF yeast strains made ORFs fused to glutathione S-transferase (GST) for ORFs fused to glutathione S-transferase (GST) for

purification purposespurification purposes Biochemical assay revealed three new Biochemical assay revealed three new

biochemical reactions associated with yeast ORFsbiochemical reactions associated with yeast ORFs

pre-tRNALigated tRNAtRNA halves

Abc1 35 64

MicrofluidicsMicrofluidics

Proteomics requires Proteomics requires greater automationgreater automation

Microfluidics: a “lab Microfluidics: a “lab on a chip”on a chip” Microvalves and Microvalves and

pumps allow control pumps allow control of nanoliter amountsof nanoliter amounts

Can control Can control biochemical biochemical reactionsreactions

A microfluidics chip

Microfluidics in actionMicrofluidics in actionloading compartmentalization

purgingmixing

500 m

Summary ISummary I Goals of proteomics Goals of proteomics

Identify and ascribe function to proteins under Identify and ascribe function to proteins under all biologically plausible conditionsall biologically plausible conditions

Proteomics methodsProteomics methods 2-D gel electrophoresis for separating proteins on the 2-D gel electrophoresis for separating proteins on the

basis of charge and molecular weightbasis of charge and molecular weight Mass spectrometry for identifying proteins by Mass spectrometry for identifying proteins by

measuring the mass-to-charge ratio of their ionized measuring the mass-to-charge ratio of their ionized peptide fragmentspeptide fragments

Protein chips to identify proteins, to detect protein–Protein chips to identify proteins, to detect protein–protein interactions, to perform biochemical assays, protein interactions, to perform biochemical assays, and to study drug–target interactionsand to study drug–target interactions

Summary IISummary II

Proteomics methods (continued)Proteomics methods (continued) Yeast two-hybrid method for studying protein–Yeast two-hybrid method for studying protein–

protein interactionsprotein interactions Biochemical genomics for high-throughput assaysBiochemical genomics for high-throughput assays

Some accomplishments of proteomicsSome accomplishments of proteomics Example: yeastExample: yeast

Yeast two-hybrid method reveals interactomeYeast two-hybrid method reveals interactome Transcriptional regulatory networks deducedTranscriptional regulatory networks deduced Biochemical genomics uncovers new ORF Biochemical genomics uncovers new ORF

functionsfunctions Subcellular localization of proteinsSubcellular localization of proteins