1. 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.

2. 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 acquisition 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.

3. Genomics DNA (Gene) Functional Genomics TranscriptomicsRNA Proteomics PROTEIN Metabolomics METABOLITE Transcription Translation Enzymatic reaction The “omics” nomenclature…

4. Why study protein expression? DNA Primary RNA transcript mRNA protein Modified protein Transcriptional control RNA Processing control RNA Transport control Inactive mRNA RNA Degradation control Translation control Post-translational control

5. 2D gel electrophoresis –Method for separating and visualizing proteins –Separation by charge and mass Mass spectrometry –High throughput analysis and identification of proteins. –Fragmentation of proteins –Analysis of peptides Book - pages 273-300.

6. The first dimension (separation by isoelectric focusing) - gel with an immobilised pH gradient - electric current causes charged proteins to move until it reaches the isoelectric point (pH gradient makes the net charge 0) What determines the charge of a protein? 2D-SDS PAGE gel

7. Isoelectric point (pI) Separation by charge: 4 5 6 7 8 9 10 Stable pH gradient High pH: protein is negatively charged Low pH: Protein is positively charged At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field.

8. The first dimension (separation by isoelectric focusing) - gel with an immobilised pH gradient - electric current causes charged proteins to move until it reaches the isoelectric point (pH gradient makes the net charge 0) The second dimension (separation by mass) -pH gel strip is loaded onto a SDS gel -SDS denatures the protein (to make movement solely dependent on mass, not shape) and eliminates charge. 2D-SDS PAGE gel

10. 2D-gel technique example

11. Advantages vs. Disadvantages Good resolution of proteins Detection of posttranslational modifications Not for hydrophobic proteins Limited by pH range Not easy for low abundant proteins Analysis and quantification are difficult

12. Current Mass Spec Technologies Proteome profiling/separation –2D SDS PAGE - identify proteins –2-D LC/LC - high throughput analysis of lysates (LC = Liquid Chromatography) –2-D LC/MS (MS= Mass spectrometry) Protein identification –Peptide mass fingerprint –Tandem Mass Spectrometry (MS/MS) Quantative proteomics –ICAT (isotope-coded affinity tag) –ITRAQ

13. Mass Spectrometry (MS) Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyzer Detect ions

14. 2D - LC/LC Study protein complexes without gel electrophoresis Peptides all bind to cation exchange column Peptides are separated by hydrophobicity on reverse phase column Successive elution with increasing salt gradients separates peptides by charge Complex mixture is simplified prior to MS/MS by 2D LC (trypsin)

15. 2D - LC/MS

16. Methods for protein identification

17. Identifying proteins Trypsin - digest your protein –Digests after R and K amino acids. Run peptide fragments on mass spec Digest the protein database “in silico” Compare mass spec data with theoretical data. What must be true to identify your protein?

18. Protein Identification by MS Artificial spectra built Artificially trypsinated Database of sequences (i.e. SwissProt) Spot removed from gel Fragmented using trypsin Spectrum of fragments generated MATCH Library

19. How protein sequencing works Use Tandem MS: two mass analyzer in series with a collision cell in between Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion Fragmentation of the peptides in the collision cell occur in a predictable fashion, mainly at the peptide bonds The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides… Ser-Glu-Leu-Ile-Arg-Trp Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu Ser-Glu-Leu-Ile Etc…

21. Advantages vs. Disadvantages Determination of MW and aa. Sequence Detection of posttranslational modifications High-throughput capability High capital costs Requires sequence databases for analysis

22. ISOTOPE-CODED AFFINITY TAG (ICAT): a quantitative method Label protein samples with heavy and light reagent Reagent contains affinity tag and heavy or light isotopes Chemically reactive group: forms a covalent bond to the protein or peptide Isotope-labeled linker: heavy or light, depending on which isotope is used Affinity tag: enables the protein or peptide bearing an ICAT to be isolated by affinity chromatography in a single step

23. Example of an ICAT Reagent Biotin Affinity tag: Binds tightly to streptavidin-agarose resin Linker: Heavy version will have deuteriums at * Light version will have hydrogens at * Reactive group: Thiol- reactive group will bind to Cys

24. How ICAT works? Proteolysis (eg trypsin) Lyse & Label MIX Affinity isolation on streptavidin beads Quantification MS Identification MS/MS 100 m/z 200400 600 0 100 550570 590 0 m/z Light Heavy NH 2 -EACDPLR- COOH

25. Advantages vs. Disadvantages Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%) Can be used on complex mixtures of proteins Cys-specific label reduces sample complexity Peptides can be sequenced directly if tandem MS-MS is used Yield and non specificity Slight chromatography differences Expensive Tag fragmentation Meaning of relative quantification information No presence of cysteine residues or not accessible by ICAT reagent