1. PROTEOMICS LECTURE

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

3. Gen Transcript Prote Metabol ~ome Sequence of a complete set of Genes Transcripts Proteins Metabolites = Gen Prote ~omics = Analysis of the Genome Proteome A few definitions…

4. Why study protein expression? (The steps of gene expression control) Nucleus Cytosol 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 (Gygi et al., Mol. Celll. Biol., 1990, p.1720-1730)

5. Applications of Proteomics Mining: identification of proteins (catalog the proteins) Protein-expression profile: identification of proteins in a particular state of the organism Protein-network mapping: protein interactions in living systems Mapping of protein modifications: how and where proteins are modified.

6. Proteins classes for Analysis Membrane Soluble proteins Nuclear Chromosome-associated Phosphorylated Glycosylated Complexes

7. General flow for proteomics analysis SEPARATION IDENTIFICATION

8. Current Proteomics Technologies Proteome profiling/separation –2D SDS PAGE (two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis) –2-D LC/LC (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)

9. 1) Sample loading 2) Remove the cover sheet from the IEF gel 3)Place the strip gel in the focusing tray 4) Place the strip on the top of the SDS-PAGE gel 2D-SDS PAGE gel

10. 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) 2D-SDS PAGE gel

11. 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.

12. 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 and linearises the protein (to make movement solely dependent on mass, not shape) 2D-SDS PAGE gel

14. 2D-gel technique example

15. 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

16. 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)

17. Polypeptides enter the column in the mobile phase… …the hydrophobic “foot” of the polypeptides adsorb to the hydrophobic (non polar) surface of the reverse-phase material (stationary phase) where they remain until… …the organic modifier concentration rises to critical concentration and desorbs the polypeptides Reverse Phase column

18. 2D - LC/MS

20. Methods for protein identification

21. Mass Spectrometry (MS) Stages 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

22. How the 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 (also phosphoester 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…

23. Isolates individual peptide fragments for 2 nd mass spec – can obtain peptide sequence Compare peptide sequence with protein databases (trypsin) Tandem Mass Spectrometry

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

26. Protein identification by Peptide Mass fingerprint Use MS to measure the masses of proteolytic peptide fragments. Identification is done by matching the measured peptide masses to corresponding peptide masses from protein or nucleotide sequence databases.

27. Mass spectrometry – method of separating molecules based on mass/charge ratio Compare peptide m/z with protein databases eg. MALDI-TOF (trypsin) Mass spectometry (MS)

28. 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

29. 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

30. 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

31. 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

32. 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