Principles of Proteomics overview Lim Wee Guan, PhD. Product Specialist Life Science Group Southeast Asia and Taiwan One Genome- Different Proteomes Life is base on proteins and their interactions Why proteome research? Genome Gene Transcriptional control RNA Degradation mRNA Degradation Pre-Protein Degradation Protein Degradation Translational control Posttranslational Modifications • Low correlation between mRNA abundance and protein level • Gene sequence does not code for post-translational modifications • Genome is static; Proteome reflects the dynamic nature of cellular processes What is proteomics? Proteomics is the study of protein characteristics and functions to obtain a global integrated view of: • normal and abnormal cellular processes • protein-protein interactions • regulatory networks The definition of all proteins of interest within a biological sample. Why study proteomics? Mining: identification of all proteins in a sample Protein Expression Profiling: identification of protein in a sample as a function of a state of the organism or cell under certain condition Protein Network Mapping: Approach to determine how proteins interact with each other in living systems. Mapping of protein modifications: identification how and where proteins are modified. The Question Cell specific expression Culture conditions Metabolic state Drugs Temperature Dysfunctions, Cancer Stress Simultaneous protein profiling to understand the function and regulation of thousands of proteins PROTEOMICS Structural Proteomics Expression Proteomics Functional Proteomics • Compares patterns of proteins expressed in a given tissue or cell under certain conditions • Quantitative analysis and identification of proteins • Ultimately maps these changes back to the underlying or corresponding gene Key concern of proteomics The Expression Proteomics Challenge is… …the complexity of a cell and the diverse nature of proteins: Number of diff proteins in cells/tissue/organism Humans have ~ 30,000 to 40,000 genes Estimated 100,000 -300,000 human protein count Dynamic range of proteins Expression levels vary Post-translational modifications Chemical diversity of protein -size, pI/charge, hydrophobicity,etc Key concern of proteomics How to reduce the complexity of samples Separate the proteins in an electrical field: electrophoresis Electrophoresis using 2 different properties By isoelectric point (pI): IEF IEF By size (MW): SDS-PAGE 2-D Electrophoresis Proteins migrate through the gel at a rate inversely proportional to their size. Smallest proteins travel the furthest distance Isoelectric Focusing (IEF) with IPG strips 70 - 2 40 m m 3 -4 m m + ge l pH 3 Pla s tic b a c k in g – pH 7.5 pH 10 – + pH 3 pH 7.5 – + pH 3 pH 7.5 pH 10 – + pH 3 pH 10 pH 7.5 pH 10 2D proteomics for maximum resolution pH 3 pH 3 pH 7.5 + pH 7.5 MW –pH10 pI – pH10 size charge Combine IEF and SDS-PAGE to get maximum separation Birth of 2D!!! What is the protein expression pattern at a particular point in time? Cell specific expression Culture conditions Metabolic state Drugs Temperature Dysfunctions, Cancer Stress Simultaneous protein profiling to understand the function and regulation of thousands of proteins Advantage of 2D • Global view of protein expression, ability to find signaling cascade. • Ability to detect PTM • Direct correlation of protein expression to disease • Quantitative analysis of protein regulation • Routinely separate 3000 spots per gel • Mass spect compatible 2-D Expression Proteomics Methodology FIRST DIMENSION 3 SAMPLE PREPARATION 4 pH 7 10 - + - + Condition A Condition B MASS SPECTROMETRIC IDENTIFICATION OF SPOTS STAINING ANALYSIS & EXTRACTION IMAGING SECOND DIMENSION 2-D Expression Proteomics Tools Sample Preparation 1st dimension IEF 2nd dimension SDS-PAGE MS Gel staining Spot excision Imaging and Analysis The next steps… Destain and Tryptic Digest • Cleave at known residues • Optimal length for MS Analysis 6-20 amino acid sequences • Widely used for database lookups Mass Spec Workflow What do scientists want from Mass Spec data? Data on: • peptide masses • peptide fragmentation What makes good data? • sensitivity • resolution • mass accuracy What does Mass Spec data look like? What do scientists do with Mass Spec data? • Peptide masses are compared with real database values • Peptide Mass Fingerprinting allows proteins to be identified for amino acid sequence • Software is Key: 1. Controlling the systems 2. Probability and Statistical Algorithms 3. Organization of Data Protein is Identified…Now What? Experimental Timeframe 2D PAGE separation, Image analysis and Protein excision 1. Rehydration of first dimension gels: 12 hours 2. Isoelectric focusing: 2 - 16 hours (depending on gel length and pI range to be analyzed) 3. Equilibration and SDS-PAGE separation: 2 – 8 hours (depending on gel size) 4. Gel fixation and staining: 4 hours to overnight 5. Image analysis: 1 – 5 days / comparison set (depending on gel number, protein number) 6. Protein excision: 3 hours – 1 day (depending on protein number) Total: 5 – 10 days Mass Spectrometry: 1. Protein digestion: 7 hours (overnight) 2. Sample concentration: 0.25 - 0.5 day 3. Sample Loading: 0.25 - 0.5 day 4. MALDI MS analysis; spectra storage: 0.75 days to overnight 5. Spectra interpretation: 1 day 6. Database submission: 1 - 4 days (manual) 7. Protein identification: 5 - 7 days. Total: 9.5- 15 days Once the protein sample has been prepared, 2D PAGE separation, MS analysis and subsequent protein identification usually takes between 15-25 days. Research Research Sample prep FIRST DIMENSION pH 3 SAMPLE PREPARATION 4 7 10 - + - + Condition A Condition B MASS SPECTROMETRIC IDENTIFICATION OF SPOTS STAINING ANALYSIS & EXTRACTION IMAGING SECOND DIMENSION What is sample prep? Sample preparation involves everything that lies between the sample and 1st dimension IEF Sample Protein mixture • Cell disruption/ lysis • Protein extraction, solubilization • Contaminant removal/ cleanup • Fractionation The Equation • Good sample preparation = = Good 2D Gels Bad sample prep = bad 2D gels Sample Preparation Tools for Sample Cleanup and Fractionation Sample Preparation Sample prep requirements • Reproducibly solubilize proteins of all classes, including hydrophobic proteins • Prevent protein aggregation and loss of solubility during isoelectric focusing • Yield proteins of interest at detectable levels, which may require the removal of interfering abundant proteins or non-relevant classes of protein 2 Sample Prep Strategies : General Purpose Cleanup • Improve Resolution • Improve Reproducibility Fractionation • Reduce Complexity • Improve Range of Detection • Enrich low-abundance proteins Applicable to most sample types, applications •Contaminant removal Salts Detergents, phenolics, etc. Abundant proteins •Disulfide bond removal (reduction/ alkylation) •Total protein extraction Contaminant removal Introduction of contaminants from…… Cell/ tissue lysis method Sample source Detergents Nucleic acids Salts Lipids, sugars Buffers Phenolics Insoluble material Ions, small molecules Abundant proteins Need for contaminant removal Problem: salt contamination Horizontal streaking caused by excess salt (lysis method) Problem: detergent contamination Horizontal streaking caused by excess SDS (lysis method) Solution: ReadyPrep 2-D Cleanup Kit Untreated Treated E. coli cell lysates spiked with 1% SDS Improved resolution, less streaking on 2-D gels Removes salts & detergents, phenolic compounds, lipids, ionic contaminants >95% recovery Concentrates dilute samples Solution: Bio-Spin columns for buffer exchange Size Exclusion Chromatography • Large molecules (proteins) pass around beads • Small molecules (salt) retained in beads Requires buffer exchange with IEF buffer Bio-Spin 6 and Micro Bio-Spin 6 • Bio-Spin: 50-100 ml sample • Micro Bio-Spin: 10-75 ml sample Problem: Albumin and IgG in serum samples Abundant proteins impair detection of other proteins Solution: Aurum Serum Protein and Affi-Gel Blue kits Affinity chromatography • Albumin and/or IgG bind to column (removed) • Other proteins pass through column • Some proteins bind to column due to non-specific protein interactions with albumin and IgG Aurum Serum Protein and Affi-Gel Blue kits Bead Problem: Disulfide bond removal • • • • Disulfide bridges within and between proteins cause spurious spot patterns Prevention: Reducing agents A big problem in the alkaline region Reducing agents tend to be negatively charged • DTT migrates off of gel during IEF Solution: Reduction followed by alkylation •Reduction with TBP •Alkylation with IAA (prevents reformation of disulfide bonds) •Prior to IEF ReadyPrep Reduction/Alkylation kit Mouse liver extract run on pH 7-10, 11 cm strips. Cup loaded. The challenge: see low expressed proteins • Estimated ~100,000 – 300,000 human protein count • Spot counts per gel typically 500 - 2000 depending on gel size and pI range • Sample load limits detection of low- abundance proteins Need for enrichment Strategy 1: Increase loading 50 µg E.Coli 500 µg E.Coli 100 µg E.Coli 1000 µg E.Coli 250 µg E.Coli Strategy 2: Increase gel size pH 5 7 cm pH 8 8% 60 µg E. coli 944 spots 11 cm 16% 100 µg E. coli 1,287 spots 150 µg E. coli 1,724 spots 17 cm Strategy 3: Narrow-range IPGs pH 3-10 pH 3- 6 pH 5- 8 pH 7-10 Strategy 4: Fractionation Separation of a protein sample into fractions according to Cellular location Solubility/ hydrophobicity Size (mw) Charge/ pI Strategy 4: Fractionation Fractionation enables analysis of components Reduced sample complexity Increased resolution (more dimensions) Increased load of proteins of interest Enrichment of low-abundance proteins Fractionation Application-dependent •Cellular location •Biophysical property •Solubility/ hydrophobicity •Size •Charge/ pI Fractionation by cellular location ReadyPrep Protein Extraction kits Cytoplasmic/Nuclear Isolates nuclei, extracts proteins Membrane I Triton X-114, temp.-dep. Partitioning Membrane II Carbonate extraction Signal Triton X-100 differential solubility Caveoli-associated proteins Membrane protein isolation Signal Membrane I Membrane II Mouse liver isolated with different kits. ~450 ug extract loaded onto 17 cm pH 3-10 IPG strips. Total Protein Fractionation by protein solubility ReadyPrep Sequential Extraction Kit sample ReadyPrep Protein Extraction Kit (Soluble/Insoluble) sample Soluble proteins Solution 1 Solution 2 Solution 3 SB 3-10 ASB14 Insoluble proteins Benefits of these approaches • Enrichment of proteins of interest • Well-understood protocols, in kit format Reproducibility Ease-of-use Easy to try a number of approaches Fractionation by size: Model 491 prep cell Effective visualization of •Protein families •Post translational modifications Fractionation by size: Bio-Spin 30 Size Exclusion Chromatography •Large molecules (proteins) pass around beads •Small molecules retained in beads Bio-Spin 6 and Micro Bio-Spin 30 • P30: >40 kDa Additional chromatographic options • Bio-Gels 2, 4, 10, 60, and 100 • Fractionation ranges from 2 -100 kDa Fractionation by charge Ion Exchange Chromatography • • • • • Positively charged proteins (basic) bind to CEX (cation exchange) Negatively charged proteins (acidic) bind to AEX (anion exchange) Elution with high salt, but pH gradient also effective High dynamic binding capacity Charge of protein is dependent on buffer conditions Aurum AEX and CEX kits pI=4 pI=9 ----- AEX --- + --- + --- CEX + + + Fractionation by charge Total Protein AEX CEX 1 Acidic protein binds to AEX not CEX 3 Basic protein binds to CEX not AEX 5 Protein is enriched on CEX 4 Protein is enriched on AEX Fractionation by pI Removes proteins outside of pI range of interest Pair with narrow- and micro-range IPGs (2DGE) All performed in liquid medium Particularly helpful with • High molecular weight proteins • Hydrophobic/ membrane proteins • Extreme pIs The Rotofor® cell Fractionation by pI MW Direct 2-DE: pH 3 - 6 pH 4 - 7 pH 5 - 8 70 50 35 30 15 8 Prefractionated 2-DE: pH 3 - 6 pH 4 - 7 pH 5 - 8 70 50 35 30 15 CSF sample pH 1.5 - 4.5 CSF sample pH 4.5-6.0 CSF sample pH 6.0 –7.5 8 Sample preparation products Sample preparation in proteomics • Critically important consideration in expression proteomics research • No single approach fits all Sample type Size, source Research goal Laboratory resources Unique position Bio-Rad has an extensive product offering for 2-D sample preparation products! First Dimension Tools ReadyPrep 2D starter kit: •Rehydration buffer •Equilibration buffer I •Equilibration buffer II •Glycerol solution •Agraose overlay •IAA Sample prep tools 2-D Electrophoresis Tools for Rapid, High-Resolution Protein Separations 2-D Electrophoresis 2-D Electrophoresis Workflow Precast Gels 2-D Tools Stains Stainers • Simplified staining ensures consistent results • Improved gel handling • Available in two sizes (small and large) • Stain compatibility Imaging and Analysis Tools for Acquisition and Analysis of Protein Expression Data PDQuest Software Imaging instruments Spot cutter instrument Imaging Detection capabilities GS-800 Molecular Imager FX Pro (Plus) Systems VersDoc Model 4000 GS-800 Densitometer VersaDoc Model 4000 Molecular Imager FX Pro-Q Diamond ● Pro-Q Emerald ● Cy5 ● y2 ● Cy3 ● SYPRO Orange ● ● SYPRO Red ● ● SYPRO Ruby ● ● Coomassie Blue ● ● ● Copper ● ● ● Silver ● ● ● Zinc ● ● ● Research Assessment of acetaminophen toxicity in mouse liver EXQuest Spot Cutter • 2-D and 1-D spot cutting •Gel matching and excision driven by PDQuest software • High speed precision robotics -Cuts 600 spots per hour -Accuracy of +0.1 mm ensures accurate excision of small, closely grouped spots •Compatible with visible light and UV-based applications, including SYPRO Ruby and Orange, silver, and Coomassie Blue • Cuts free or backed gels and PVDF membranes 2-D Expression Proteomics Tools Sample Preparation 1st dimension IEF 2nd dimension SDS-PAGE MS Gel staining Spot excision Imaging and Analysis Support www.expressionproteomics.com Integration with PDQuest •PDQuest controls the imaging instruments •PDQuest optimizes/normalizes images for accurate comparison •Analytical tools identify protein spots of interest •Advanced algorithms identify & match protein patterns •Controls the ProteomeWorks Plus spot cutter •Stores, manages and records sample info Discover with Bio-Rad! • Sample Preparation Large and small scale Electrophoretic, chromatographic, or chemical reagent based Most complete line of sample prep devices and kits for proteomics available • Electrophoresis 7, 11, 17, 18, and 24 cm IPG strips for first dimension Broad, non linear, narrow and micro pH range Mini, midi, and large format precast gels and gel rigs for second dimension Complete line of power supplies and reagents • Image Analysis Densitometer, laser based, and a complete line of camera based imagers for every need • PDQuest 2-D Analysis Software Functionality for all 2-D applications Over 25 years of PDQuest 2-D Analysis Software assisted discoveries 300+ publications since 1976 Over 100 from 2002-present • Spot cutting Fastest and most accurate instrument available Integrated with full suite of expression proteomics products Thank you