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Proteomics

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Next week - structural proteomics, protein:protein interactions, subcellular localization ... Major cause of nosocomial infections. ... – PowerPoint PPT presentation

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Title: Proteomics


1
Proteomics
  • Handout
  • Another on the website - copier not cooperating.
  • Today - overview
  • Thursday - Debora - tools
  • Next week - structural proteomics,
    proteinprotein interactions, subcellular
    localization
  • Reminder - Exam - March 4

2
Genome of the week - Enterococcus faecalis
  • E. faecalis - urinary tract infections,
    bacteremia, endocarditis.
  • Organism sequenced is vancomycin resistant.
  • Vancomycin is often last available antibiotic -
    resistance to this drug often means no other
    antibiotics will work.
  • Major cause of nosocomial infections.
  • Possible transfer of vanomycin resistance genes
    to more serious pathogens is a major concern.

3
Genome of the week - Enterococcus faecalis
  • Over 25 of the E. faecalis genome consists of
    foreign DNA.
  • Phages, insertions sequences, transposons.
  • Likely contributed to the acquistion of
    resistance to multiple antibiotics.
  • Over 35 PTS systems
  • Responsible for transporting sugars into the
    cell.
  • Most found in any sequenced genome, likely
    utilize undigested sugars in the intestine.

4
Why study proteins?
  • They are the machines that make cells function.
  • RNA levels do not always accurately predict
    protein levels.
  • Often processes are regulated at the
    transcriptional level.
  • Some processes are controlled post-transcriptional
    ly.
  • Most often proteins are the targets of drugs.

5
Proteomics -large scale analysis of proteins
  • Protein levels - Determining the abundance of
    proteins in a sample.
  • 2D gel electrophoresis, mass spectrometry,
    protein microarrays
  • Interacting proteins - determining which proteins
    come together to form functional complexes.
  • Yeast 2-hybrid, affinity purification
  • Subcellular localization - site of localization
    can often provide clues to the function of a
    protein.
  • GFP tagging, immunofluorescence microscopy.
  • Protein activity - investigating the biochemical
    activities of proteins.
  • Structural genomics - high-throughput analysis of
    the protein structure

6
From www.probes.com
7
Proteins
  • Primary structure - sequence
  • Searching databases
  • Identifying functional domains
  • Secondary and tertiary structure - 3D folding of
    proteins.
  • Proteins have unique 3D structures
  • Identify functional domains
  • VAST - online structural tool from NCBI

8
Western Blot
  • Determine the presence and level of a protein in
    a cell lysate.
  • http//web.mit.edu/esgbio/www/rdna/rdna.html -
    review of Northern, Western, and Southern blots.

9
Monitoring protein levels - large scale
  • 2D gel electrophoresis
  • Old technology - not as useful for lowly
    expressed proteins.
  • Mass spectrometry
  • Many new techniques for protein detection and
    quantitation being developed.
  • Protein microarrays
  • Many developing technologies

10
Protein microarrays
  • Analysis of thousands of proteins at one time.
  • Many different types
  • Antibody arrayed - detect many proteins
  • Proteins arrayed - detect interacting proteins
  • Proteins arrayed - detect interacting small
    molecules
  • Etc.

11
Templin et al. 2002 Trend in Biotch. Vol 20
12
Proteinprotein interactions
13
Protein activity arrays
14
Small molecule arrays
15
From the Macbeath laboratory website. See for
more info the following website http//cgr.harvar
d.edu/macbeath/research/protein_microarrays/protei
n_microarrays.html
16
Why bother with DNA microarrays?
  • Protein microarrays are not as robust
  • DNA is DNA - all features will behave similarly
    under single hybridization conditions.
  • Proteins are unique - will behave differently.
  • Protein microarrays are costly
  • 500-1000 per antibody
  • 10 per oligo
  • Used for different purposes
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