Sample Quality is Key Garbage in Garbage out

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

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

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

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

Narrow Range IEF (“Zoom”)

$Pre-fractionation: Subcellular Fractionation (sucrose gradient)$

Pre-fractionation: Subcellular Fractionation (sucrose gradient)

$Pre-fractionation: Laser Capture Micro-dissection Isolation of individual Purkinje Cells Isolation of intestinal cell types$

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

$Pre-fractionation: IEF (Isoelectric point)$

Pre-fractionation: IEF (Isoelectric point)

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

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

$Pre-fractionation: Immuno-depletion$

Pre-fractionation: Immuno-depletion

$Pre-fractionation: Immuno-depletion$

Pre-fractionation: Immuno-depletion

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

$EXPRESSION PROTEOMICS ANCILLARY TECHNIQUES Subcellular fractionation Protein depletion Affinity capture for protein: protein interactions$

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

$EXPRESSION PROTEOMICS Pre-fractionation of sample; Simplify mixture More complex sample = crowded gel pattern;$

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

$EXPRESSION PROTEOMICS Pre-fractionation of sample; Simplify mixture Membrane isolation (equine sperm) I II Ultracentrifuge$

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

$EXPRESSION PROTEOMICS Pre-fractionation of sample; Simplify mixture Nucleoli Isolation Ref: Directed Proteomic Analysis of$

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 Current Biology (2002), 12, 1 -11.

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

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