protein-protein interactions “the interactome” yeast two-hybrid analysis yeast two-hybrid...
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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
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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
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A schematic of the yeast two-A schematic of the yeast two-hybrid methodhybrid method
m
n
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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TAP TAP Purification Purification
StrategyStrategy
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Identification of Interacting Identification of Interacting ProteinsProteins
ProteolyticDigestion(Trypsin)
MassSpectrometric
Analysis
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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
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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
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Principle of mass Principle of mass spectrometryspectrometry
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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
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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
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A mass spectrumA mass spectrum
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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
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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
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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)
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Enzymatic Enzymatic ComplementationComplementation
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Blue=DAPI
Red=BGAL
Blue=DAPI
Green=BGAL
Red=Actin
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Bimolecular Fluorescence Bimolecular Fluorescence ComplementationComplementation
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FRET (fluorescence resonance FRET (fluorescence resonance energy transfer)energy transfer)
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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”
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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
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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
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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
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Microfluidics in actionMicrofluidics in actionloading compartmentalization
purgingmixing
500 m
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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
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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