Proteomics • Handout • Another on the website - copier not cooperating. • Today - overview • Thursday - Debora - tools • Next week - structural proteomics, protein:protein interactions, subcellular localization • Reminder - Exam - March 4
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.
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.
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-transcriptionally. • Most often proteins are the targets of drugs.
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
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
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.
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
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.
From the Macbeath laboratory website. See for more info the following website: http://cgr.harvard.edu/macbeath/research/protein_microarrays/protein_microarrays.html
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