Genomics II:The Proteome

Using high-throughput methods to identify proteins and to

understand their function

Subcellular localization of the yeast proteome

• Complete genome sequences allow each ORF to be precisely tagged with a reporter molecule

• Tagged ORF proteins indicate subcellular localization– Useful for the following:

• Correlating to regulatory modules• Verifying data on protein–protein interactions• Annotating genome sequence

Attaching a GFP tag to an ORF

Fusion protein

Chromosome

PCR product

COOHNH2

Homologousrecombination

GFP HIS3MX6

ORF1 ORF2

protein GFP

FlyTrap Screen for

Protein Localization

http://flytrap.med.yale.edu/

Patterns of protein localization

Distribution of subcellular localization

Identification of unpredicted ORFs

Protein-protein interactions“The Interactome”

• Yeast two-hybrid analysis

• Protein chips

• Biochemical purification/Mass spectrometry

• Protein complementation

Yeast two-hybrid method• Goal: Determine how proteins interact with each other

• Method– Use yeast transcription factors

– Gene expression requires the following:• A DNA-binding domain• An activation domain• A basic transcription apparatus

– Attach protein1 to DNA-binding domain (bait)

– Attach protein2 to activation domain (prey)

– Reporter gene expressed only if protein1 and protein2 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– 5,345 were successfully cloned into yeast as

both bait and prey– Identity of ORFs determined by DNA

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

Yeast two-hybrid results for flies & worms

• Worms:– Created >3000 bait constructs– Tested against two AD libraries– Mapped 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 yeast two-hybrid method

• There is evidence that other methods may be more sensitive

• Some inaccuracy reported when compared against known protein–protein interactions– False positives– False negatives

Purification of interacting proteins

• Immunoprecipitation– Impractical on large scale (identification of

unknowns)

• Affinity purification– Biochemically practical, but too dirty

• Tandem affinity purification– Sufficient yield & purity for identification of

unknown proteins

TAP Purification

Strategy

Identification of Interacting Proteins

ProteolyticDigestion(Trypsin)

MassSpectrometricAnalysis

Identifying proteins with mass spectrometry

• Preparation of protein sample– Extraction from a gel– Digestion by proteases — e.g., trypsin

• Mass spectrometer measures mass-charge ratio of peptide fragments

• Identified peptides are compared with 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

Mass spectrometry

• Measures mass-to-charge ratio

• Components of mass spectrometer– Ion source– Mass analyzer– Ion detector– Data acquisition unit

A mass spectrometer

Principle of mass spectrometry

Ion sources used for proteomics

• Proteomics requires specialized ion sources

• Electrospray Ionization (ESI)– With capillary

electrophoresis and liquid chromatography

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

surface

ESI

MALDI

Mass analyzers used for proteomics

• Ion trap– Captures ions on the basis

of mass-to-charge ratio– Often used with ESI

• Time of flight (TOF)– Time for accelerated ion to

reach detector indicates mass-to-charge ratio

– Frequently used with MALDI

• Also other possibilities

Ion Trap

Time of Flight

Detector

A mass spectrum

Identifying proteins with mass spectrometry

• Preparation of protein sample– Extraction from a gel– Digestion by proteases — e.g., trypsin

• Mass spectrometer measures mass-charge ratio of peptide fragments

• Identified peptides are compared with 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

Limitations of mass spectrometry

• Not very good at identifying minute quantities of protein

• Trouble dealing with phosphorylated proteins

• Doesn’t provide concentrations of proteins

• Improved software eliminating human analysis is necessary for high-throughput projects