Protein-protein interactions “The Interactome” Yeast two-hybrid analysis Yeast two-hybrid analysis Protein chips Protein chips Biochemical purification/Mass spectrometry Biochemical purification/Mass spectrometry

Yeast two-hybrid method Goal: Determine how proteins interact with each other Goal: Determine how proteins interact with each other Method Method Use yeast transcription factors Use yeast transcription factors Gene expression requires the following: Gene expression requires the following: A DNA-binding domain A DNA-binding domain An activation domain An activation domain A basic transcription apparatus A basic transcription apparatus Attach protein 1 to DNA-binding domain (bait) Attach protein 1 to DNA-binding domain (bait) Attach protein 2 to activation domain (prey) Attach protein 2 to activation domain (prey) Reporter gene expressed only if protein 1 and protein 2 interact with each other Reporter gene expressed only if protein 1 and protein 2 interact with each other

A schematic of the yeast two-hybrid method m n

Results from a yeast two-hybrid experiment Goal: To characterize protein–protein interactions among 6,144 yeast ORFs Goal: To characterize protein–protein interactions among 6,144 yeast ORFs 5,345 were successfully cloned into yeast as both bait and prey 5,345 were successfully cloned into yeast as both bait and prey Identity of ORFs determined by DNA sequencing in hybrid yeast Identity of ORFs determined by DNA sequencing in hybrid yeast 692 protein–protein interaction pairs 692 protein–protein interaction pairs Interactions involved 817 ORFs Interactions involved 817 ORFs

Yeast two-hybrid results for flies & worms Worms: Worms: Created >3000 bait constructs Created >3000 bait constructs Tested against two AD libraries Tested against two AD libraries Mapped 4000 interactions Mapped 4000 interactions Flies: Flies: Screened 10,000 predicted transcripts Screened 10,000 predicted transcripts Found 20,000 interactions Found 20,000 interactions Statistically assigned 4800 as “high quality” interactions Statistically assigned 4800 as “high quality” interactions

Caveats associated with the yeast two-hybrid method There is evidence that other methods may be more sensitive There is evidence that other methods may be more sensitive Some inaccuracy reported when compared against known protein–protein interactions Some inaccuracy reported when compared against known protein–protein interactions False positives False positives False negatives False negatives

Protein chips Thousands of proteins analyzed simultaneously Thousands of proteins analyzed simultaneously Wide variety of assays Wide variety of assays Antibody–antigen Antibody–antigen Enzyme–substrate Enzyme–substrate Protein–small molecule Protein–small molecule Protein–nucleic acid Protein–nucleic acid Protein–protein Protein–protein Protein–lipid Protein–lipid Yeast proteins detected using antibodies

Fabricating protein chips Protein substrates Protein substrates Polyacrylamide or agarose gels Polyacrylamide or agarose gels Glass Glass Nanowells Nanowells Proteins deposited on chip surface by robots Proteins deposited on chip surface by robots

Protein attachment strategies Diffusion Diffusion Protein suspended in random orientation, but presumably active Protein suspended in random orientation, but presumably active Adsorption/Absorption Adsorption/Absorption Some proteins inactive Some proteins inactive Covalent attachment Covalent attachment Some proteins inactive Some proteins inactive Affinity Affinity Orientation of protein precisely controlled Orientation of protein precisely controlled Diffusion Adsorption/ Absorption Covalent Affinity

Classes of capture molecules Different capture molecules must be used to study different interactions Different capture molecules must be used to study different interactions Examples Examples Antibodies (or antigens) for detection Antibodies (or antigens) for detection Proteins for protein- protein interaction Proteins for protein- protein interaction Enzyme-substrate for biochemical function Enzyme-substrate for biochemical function Receptor– ligand Antigen– antibody Protein– protein Aptamers Enzyme– substrate

Reading out results Fluorescence Fluorescence Most common method Most common method Fluorescent probe or tag Fluorescent probe or tag Can be read out using standard nucleic acid microarray technology Can be read out using standard nucleic acid microarray technology Surface-enhanced laser desorption/ionization (SELDI) Surface-enhanced laser desorption/ionization (SELDI) Laser ionizes proteins captured by chip Laser ionizes proteins captured by chip Mass spectrometer analyzes peptide fragments Mass spectrometer analyzes peptide fragments Atomic-force microscopy Atomic-force microscopy Detects changes in chip surface due to captured proteins Detects changes in chip surface due to captured proteins

Difficulties in designing protein chips Unique process is necessary for constructing each probe element Unique process is necessary for constructing each probe element Challenging to produce and purify each protein on chip Challenging to produce and purify each protein on chip Proteins can be hydrophobic or hydrophilic Proteins can be hydrophobic or hydrophilic Difficult to design a chip that can detect both Difficult to design a chip that can detect both

Purification of interacting proteins Immunoprecipitation Immunoprecipitation Impractical on large scale (identification of unknowns) Impractical on large scale (identification of unknowns) Affinity purification Affinity purification Biochemically practical, but too dirty Biochemically practical, but too dirty Tandem affinity purification Tandem affinity purification Sufficient yield & purity for identification of unknown proteins Sufficient yield & purity for identification of unknown proteins

TAP Purification Strategy

Identification of Interacting Proteins Proteolytic Digestion (Trypsin) Mass Spectrometric Analysis

Identifying proteins with mass spectrometry Preparation of protein sample Preparation of protein sample Extraction from a gel Extraction from a gel Digestion by proteases — e.g., trypsin Digestion by proteases — e.g., trypsin Mass spectrometer measures mass-charge ratio of peptide fragments Mass spectrometer measures mass-charge ratio of peptide fragments Identified peptides are compared with database Identified peptides are compared with database Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database Match of experimental readout to database PMF allows researchers to identify the protein Match of experimental readout to database PMF allows researchers to identify the protein

Mass spectrometry Measures mass-to- charge ratio Measures mass-to- charge ratio Components of mass spectrometer Components of mass spectrometer Ion source Ion source Mass analyzer Mass analyzer Ion detector Ion detector Data acquisition unit Data acquisition unit A mass spectrometer

Principle of mass spectrometry

Ion sources used for proteomics Proteomics requires specialized ion sources Proteomics requires specialized ion sources Electrospray Ionization (ESI) Electrospray Ionization (ESI) With capillary electrophoresis and liquid chromatography With capillary electrophoresis and liquid chromatography Matrix-assisted laser desorption/ionization (MALDI) Matrix-assisted laser desorption/ionization (MALDI) Extracts ions from sample surface Extracts ions from sample surface ESI MALDI

Mass analyzers used for proteomics Ion trap Ion trap Captures ions on the basis of mass-to-charge ratio Captures ions on the basis of mass-to-charge ratio Often used with ESI Often used with ESI Time of flight (TOF) Time of flight (TOF) Time for accelerated ion to reach detector indicates mass-to-charge ratio Time for accelerated ion to reach detector indicates mass-to-charge ratio Frequently used with MALDI Frequently used with MALDI Also other possibilities Also other possibilities Ion Trap Time of Flight Detector

A mass spectrum

Identifying proteins with mass spectrometry Preparation of protein sample Preparation of protein sample Extraction from a gel Extraction from a gel Digestion by proteases — e.g., trypsin Digestion by proteases — e.g., trypsin Mass spectrometer measures mass-charge ratio of peptide fragments Mass spectrometer measures mass-charge ratio of peptide fragments Identified peptides are compared with database Identified peptides are compared with database Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database Match of experimental readout to database PMF allows researchers to identify the protein Match of experimental readout to database PMF allows researchers to identify the protein

Limitations of mass spectrometry Not very good at identifying minute quantities of protein Not very good at identifying minute quantities of protein Trouble dealing with phosphorylated proteins Trouble dealing with phosphorylated proteins Doesn’t provide concentrations of proteins Doesn’t provide concentrations of proteins Improved software eliminating human analysis is necessary for high-throughput projects Improved software eliminating human analysis is necessary for high-throughput projects

Protein Complementation Enzymatic complementation Enzymatic complementation  -galactosidase reconstitution  -galactosidase reconstitution Fluorescence complementation Fluorescence complementation GFP or YFP reconstitution GFP or YFP reconstitution FRET (fluorescence resonance energy transfer) FRET (fluorescence resonance energy transfer)

Enzymatic Complementation

Blue=DAPI Red=BGAL Blue=DAPI Green=BGAL Red=Actin

Bimolecular Fluorescence Complementation

FRET (fluorescence resonance energy transfer)

Pregenomics biochemical assays Methods used to find genes responsible for specific biochemical activity before the inception of genomics Methods used to find genes responsible for specific biochemical activity before the inception of genomics Laboriously purify responsible protein Laboriously purify responsible protein Often expensive and time consuming Often expensive and time consuming Expression cloning Expression cloning Introduce cDNA pools into cells Introduce cDNA pools into cells Look for biochemical activity in those cells Look for biochemical activity in those cells Caveat: Often difficult to detect biochemical activity in cell’s biochemical “background” Caveat: Often difficult to detect biochemical activity in cell’s biochemical “background”

Biochemical genomics ( “Enzomics”? ) Genome of an organism is already known Genome of an organism is already known Approach Approach Construct plasmids for all ORFs Construct plasmids for all ORFs Attach ORFs to sequence that will facilitate purification Attach ORFs to sequence that will facilitate purification Transform cells Transform cells Isolate ORF products Isolate ORF products Test for biochemical activity Test for biochemical activity

Biochemical genomics in yeast 6,144 ORF yeast strains made 6,144 ORF yeast strains made ORFs fused to glutathione S-transferase (GST) for purification purposes ORFs fused to glutathione S-transferase (GST) for purification purposes Biochemical assay revealed three new biochemical reactions associated with yeast ORFs Biochemical assay revealed three new biochemical reactions associated with yeast ORFs pre-tRNA Ligated tRNA tRNA halves Abc1 35  64

Microfluidics Proteomics requires greater automation Proteomics requires greater automation Microfluidics: a “lab on a chip” Microfluidics: a “lab on a chip” Microvalves and pumps allow control of nanoliter amounts Microvalves and pumps allow control of nanoliter amounts Can control biochemical reactions Can control biochemical reactions A microfluidics chip

Microfluidics in action loading compartmentalization purging mixing 500  m

Summary I Goals of proteomics Goals of proteomics Identify and ascribe function to proteins under all biologically plausible conditions Identify and ascribe function to proteins under all biologically plausible conditions Proteomics methods Proteomics methods 2-D gel electrophoresis for separating proteins on the basis of charge and molecular weight 2-D gel electrophoresis for separating proteins on the basis of charge and molecular weight Mass spectrometry for identifying proteins by measuring the mass-to-charge ratio of their ionized peptide fragments Mass spectrometry for identifying proteins by measuring the mass-to-charge ratio of their ionized peptide fragments Protein chips to identify proteins, to detect protein– protein interactions, to perform biochemical assays, and to study drug–target interactions Protein chips to identify proteins, to detect protein– protein interactions, to perform biochemical assays, and to study drug–target interactions

Summary II Proteomics methods (continued) Proteomics methods (continued) Yeast two-hybrid method for studying protein–protein interactions Yeast two-hybrid method for studying protein–protein interactions Biochemical genomics for high-throughput assays Biochemical genomics for high-throughput assays Some accomplishments of proteomics Some accomplishments of proteomics Example: yeast Example: yeast Yeast two-hybrid method reveals interactome Yeast two-hybrid method reveals interactome Transcriptional regulatory networks deduced Transcriptional regulatory networks deduced Biochemical genomics uncovers new ORF functions Biochemical genomics uncovers new ORF functions Subcellular localization of proteins Subcellular localization of proteins