1. Sample Quality is Key Garbage in……………………………..Garbage out.

2. REQUIRED: GOOD PROTEIN EXTRACTION Cell Disruption Grinding/homogenizatio/pressure bombs Lyse with detergents Freeze-thaw Disable Proteinases Inhibitor cocktails Denaturants (urea, guanidine, SDS) Disrupt Nucleic Acids Nucleases Reduce & Alkylate proteins DTT or mercaptoethanol Iodoacetamide Acrylamide

3. OPTIONAL: PROTEIN DESALTING & CONCENTRATION Diminish contaminant salts & other constituents (glycerol, Detergents, alkylating agents) Concentrate sample Lyophilize Precipitate SpeedVac MWCO spin concentrators

4. PROTEIN DETECTION  Detect proteins (must be mass spec ‘friendly’)  Coomassie Brilliant Blue R-250 staining (detection limit ~100 ngm; low pmol)  Coomassie Brilliant Blue G-250 staining (detection limit ~50 ngm; low pmol)  Silver staining (non-fixing; NO GLUTARALDEHYDE; detection limit ~ 1 ngm; fmol)  Sypro  stains * (fluorescent; detection limit ~ 1 ngm; fmol) * Requires fluorescent scanner

5. Narrow Range IEF (“Zoom”)

6. Pre-fractionation: Subcellular Fractionation (sucrose gradient)

7. Isolation of individual Purkinje CellsIsolation of intestinal cell types 2D Gel MFP ID Pre-fractionation: Laser Capture Micro-dissection

8. Pre-fractionation: IEF (Isoelectric point)

9. Pre-fractionation: Molecular Weight Fractionation (Spin Concentrator) 2D Gel

10. Pre-fractionation: Immuno-depletion

12. PLAN A STRATEGY FOR PROTEOMIC ANALYSIS Tissue or Whole cell (Animal, plant, bacterial, yeast) Subcellular fractionation Protein Extraction Lysis; mechanical, enzymatic Localization Cytoskeletal Nuclear Membrane Cytosolic Finally… Concentration Complexity Buffer constituents Contaminants Species

13. EXPRESSION PROTEOMICS ANCILLARY TECHNIQUES Subcellular fractionation Protein depletion Affinity capture for protein:protein interactions 2D gel image analysis Differential protein labeling and quantitative image analysis; DIGE FUNCTIONAL PROTEOMICS iCAT & iTRAQ; quantitative mass spectrometry

14. EXPRESSION PROTEOMICS Pre-fractionation of sample; Simplify mixture More complex sample = crowded gel pattern; lower resolution Less complex sample = less crowded pattern; higher resolution Pre-fractionate samples Tissue fractionation Cell type Subcellular fractionation Membrane vs. cytosol Mitochondria Nuclei Ribosomes Fluid Protein depletion

15. EXPRESSION PROTEOMICS Pre-fractionation of sample; Simplify mixture Membrane isolation (equine sperm) SDS PAGE Before After Ultracentrifuge spin Before After I II I II MW

16. EXPRESSION PROTEOMICS Pre-fractionation of sample; Simplify mixture Nucleoli Isolation Ref: Directed Proteomic Analysis of the Human Nucleolus Jens S. Andersen, Carol E. Lyon, Archa H. Fox, Anthony K. L. Leung, Yun Wah Lam, Hanno Steen 1, Matthias Mann and Angus I. Lamond 1 Current Biology (2002), 12, 1-11.

17. EXPRESSION PROTEOMICS Protein Depletion; Simplify sample Serum depletion 85% of serum is Made up of 6 proteins Difficult to see minor proteins Photo courtesy of Genway Biotech, San Diego, CA

18. EXPRESSION PROTEOMICS Protein Depletion; Simplify sample Serum depletion Improvement: Load more protein See more minor proteins Photo courtesy of Genway Biotech, San Diego, CA

19. TANDEM-AFFINITY PURIFICATION (TAP)

20. Eur. J. Biochem. 270, 570–578 (2003) FEBS 2003

21. TANDEM-AFFINITY PURIFICATION (TAP) Eur. J. Biochem. 270, 570–578 (2003) FEBS 2003

22. EXPRESSION PROTEOMICS Simple Mini Systems for Fractionation and 2D’s Photo courtesy of Invitrogen