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Genomics II: The Proteome

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False negatives are especially a problem when the proteins being studied are membrane bound or require ... to identify proteins. * A mass-spectrometry experiment can ... – PowerPoint PPT presentation

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Title: Genomics II: The Proteome


1
Genomics IIThe Proteome
  • Using high-throughput methods to identify
    proteins and to understand their function

2
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 proteinprotein interactions
  • Annotating genome sequence

3
Attaching a GFP tag to an ORF
GFP
HIS3MX6
PCR product
Homologous recombination
Chromosome
ORF1
ORF2
protein
COOH
NH2
GFP
Fusion protein
4
FlyTrap Screen for Protein Localization
  • http//flytrap.med.yale.edu/

5
Patterns of protein localization
6
Distribution of subcellular localization
7
Identification of unpredicted ORFs
8
Protein-protein interactionsThe Interactome
  • Yeast two-hybrid analysis
  • Protein chips
  • Biochemical purification/Mass spectrometry
  • Protein complementation

9
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

10
A schematic of the yeast two-hybrid method
m
n
11
Results from a yeast two-hybrid experiment
  • Goal To characterize proteinprotein
    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 proteinprotein interaction pairs
  • Interactions involved 817 ORFs

12
Yeast two-hybrid results for flies worms
  • Worms
  • Created gt3000 bait constructs
  • Tested against two AD libraries
  • Mapped 4000 interactions
  • Flies
  • Screened 10,000 predicted transcripts
  • Found 20,000 interactions
  • Statistically assigned 4800 as high quality
    interactions

13
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 proteinprotein interactions
  • False positives
  • False negatives

14
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

15
TAP Purification Strategy
16
Identification of Interacting Proteins
Proteolytic Digestion (Trypsin)
Mass Spectrometric Analysis
17
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

18
Mass spectrometry
  • Measures mass-to-charge ratio
  • Components of mass spectrometer
  • Ion source
  • Mass analyzer
  • Ion detector
  • Data acquisition unit

A mass spectrometer
19
Principle of mass spectrometry
20
Ion sources used for proteomics
ESI
  • 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

MALDI
21
Mass analyzers used for proteomics
Ion Trap
  • 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

Time of Flight
Detector
22
A mass spectrum
23
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

24
Limitations of mass spectrometry
  • Not very good at identifying minute quantities of
    protein
  • Trouble dealing with phosphorylated proteins
  • Doesnt provide concentrations of proteins
  • Improved software eliminating human analysis is
    necessary for high-throughput projects
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