Proteomics

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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

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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.

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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.

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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.

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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

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From www.probes.com
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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

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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.

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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

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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.

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Templin et al. 2002 Trend in Biotch. Vol 20
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Proteinprotein interactions
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Protein activity arrays
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Small molecule arrays
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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
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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