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

Sample Quality is KeySample Quality is Key

Garbage in……………………………..Garbage out.

REQUIRED: GOOD PROTEIN EXTRACTIONREQUIRED: GOOD PROTEIN EXTRACTION

Cell DisruptionGrinding/homogenizatio/pressure bombsLyse with detergentsFreeze-thaw

Disable ProteinasesInhibitor cocktailsDenaturants (urea, guanidine, SDS)

Disrupt Nucleic AcidsNucleases

Reduce & Alkylate proteinsDTT or mercaptoethanolIodoacetamideAcrylamide

OPTIONAL:OPTIONAL:PROTEIN DESALTING & PROTEIN DESALTING & CONCENTRATIONCONCENTRATION

Diminish contaminant salts & other constituents (glycerol,Detergents, alkylating agents)

Concentrate sampleLyophilizePrecipitateSpeedVacMWCO spin concentrators

PROTEIN DETECTIONPROTEIN DETECTION

Detect proteins (must be mass spec ‘friendly’)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”)Narrow Range IEF (“Zoom”)

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

Isolation of individual Purkinje Cells Isolation of intestinal cell types

2D Gel MFPID

Pre-fractionation: Laser CapturePre-fractionation: Laser CaptureMicro-dissectionMicro-dissection

Pre-fractionation: IEF (Isoelectric point)Pre-fractionation: IEF (Isoelectric point)

Pre-fractionation: Molecular Weight Pre-fractionation: Molecular Weight FractionationFractionation

(Spin Concentrator)(Spin Concentrator)

2D Gel

Pre-fractionation: Immuno-depletionPre-fractionation: Immuno-depletion

PLAN A STRATEGY FOR PLAN A STRATEGY FOR PROTEOMIC ANALYSISPROTEOMIC ANALYSIS

Tissue or Whole cell (Animal, plant, bacterial, yeast)Subcellular fractionationProtein Extraction

Lysis; mechanical, enzymaticLocalization

CytoskeletalNuclearMembraneCytosolicFinally…ConcentrationComplexityBuffer constituentsContaminantsSpecies

EXPRESSION PROTEOMICSEXPRESSION PROTEOMICS

ANCILLARY TECHNIQUES

Subcellular fractionationProtein depletionAffinity capture for protein:protein interactions2D gel image analysisDifferential protein labeling and quantitative image analysis; DIGE

FUNCTIONAL PROTEOMICS

iCAT & iTRAQ; quantitative mass spectrometry

EXPRESSION PROTEOMICSEXPRESSION PROTEOMICSPre-fractionation of sample; Simplify Pre-fractionation of sample; Simplify

mixturemixtureMore complex sample = crowded gel pattern; lower resolutionLess complex sample = less crowded pattern; higher resolution

Pre-fractionate samples

Tissue fractionationCell type

Subcellular fractionationMembrane vs. cytosolMitochondriaNucleiRibosomes

FluidProtein depletion


mixturemixtureMembrane isolation (equine sperm)

SDS PAGE

Before After

Ultracentrifuge spinBefore After

I

II

I II MW


mixturemixtureNucleoli 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 Steen1, Matthias Mann and Angus I. LamondCurrent Biology (2002), 12, 1-11.

EXPRESSION PROTEOMICSEXPRESSION PROTEOMICSProtein Depletion; Simplify sampleProtein Depletion; Simplify sample

Serum depletion

85% of serum isMade up of 6 proteins

Difficult to see minor proteins

Photo courtesy of Genway Biotech, San Diego, CA

EXPRESSION PROTEOMICSEXPRESSION PROTEOMICSProtein Depletion; Simplify sampleProtein Depletion; Simplify sample

Serum depletion

Improvement:

Load more proteinSee more minor proteins

Photo courtesy of Genway Biotech, San Diego, CA

TANDEM-AFFINITY PURIFICATION TANDEM-AFFINITY PURIFICATION (TAP)(TAP)

TANDEM-AFFINITY PURIFICATION TANDEM-AFFINITY PURIFICATION (TAP)(TAP)

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

EXPRESSION PROTEOMICSEXPRESSION PROTEOMICSSimple Mini Systems for Fractionation Simple Mini Systems for Fractionation

and 2D’sand 2D’s

Photo courtesy of Invitrogen

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

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