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CEG Protein Analysis Workshop

CEG Protein Analysis Workshop. Two-Dimensional Gel Electrophoresis Yu Liang, Ph.D. Proteomics Core Facility at UC Medical Center. From Genotype to Phenotype. Genome: DNAs Transcriptome: RNAs Proteome: Proteins Physiome: Metabolites Biome: Environment. Beyond the Genome.

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CEG Protein Analysis Workshop

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  1. CEG Protein Analysis Workshop Two-Dimensional Gel Electrophoresis Yu Liang, Ph.D. Proteomics Core Facility at UC Medical Center

  2. From Genotype to Phenotype • Genome: DNAs • Transcriptome: RNAs • Proteome: Proteins • Physiome: Metabolites • Biome: Environment

  3. Beyond the Genome • Proteins are ultimately responsible for all biological processes that take place within cells. • Protein dynamics reflect the state of biological system at a given time • Detection and identification of post-translational modifications (PTM)

  4. Proteomics Overview Spot Detection & Image Analysis Sample Preparation 2-D Electrophoresis (I) (II) (III) Enzymatic Digestion Peptide-Mass Fingerprinting Protein Identification Peptide Sequencing via MS Database Search (IV) (V) (VI)

  5. Personnel • Yu Liang, Ph.D. Research Associate MSB 5301 Tel: 558-2347 liangyu@ucmail.uc.edu http://www.med.uc.edu/proteomics/ • John Maggio, Ph.D. Professor and Chair Department of Pharmacology & Cell Biophysics

  6. Proteomics Core Equipment • Genomic Solutions Investigator 2-D Electrophoresis System • Genomic Solutions Gel Casting System • Genomic Solutions ProImage Image Acquisition System • Dell Optiplex Image Analysis Computers • New: • Fuji Fluorescent Image Analyzer FLA5100 (Phosphoprotein staining and DIGE)

  7. Genomic Solutions 2D Electrophoresis System pH phaser isoelectric focusing system Investigator 2-D electrophoresis running system

  8. 2-D Image (Fluorescent Staining) • mouse cardiac • 250 g loading • pH 3-10 IEF strips • 12.5% SDS-PAGE • File ID: mb699

  9. Customized Core Services • Complete 2-D Gel Service with Spot Picking for MS Analysis • Complete 2-D Gel Service, or (1) IEF Only (2) SDS-PAGE (large format) Only (3) SDS-PAGE Plus (staining & imaging) (4) Gel Staining (fluorescent, Sypro Ruby) (5) Image Analysis Only (6) Image Analysis Plus (spot pick) • Two new services: (1) Phosphoprotein staining by Pro-Q Diamond fluorescent dye (2) Difference gel electrophoresis (DIGE) by Cydye labelling

  10. Sample gels Control Experimental

  11. 2-D Image Control Experimental • mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso; spot ID: S8-1

  12. Spot ID: S8-1 2-D Image Experimental Control • mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso

  13. PTM? Downregulation? 2-D Image Control Experimental • mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc5bcon vs. sc15iso

  14. 2-D Gel Analysis

  15. 2-D Gel Analysis

  16. 2-D Gel Analysis

  17. Deliverables • Fluorescence-stained 2-D gels • Electronic image files • Spot list • Normalized volumes of spots Plus • Free access to our software for further analysis • Free advice and consultation on further protein characterization as well as 2-D image analysis

  18. www.med.uc.edu/proteomics

  19. www.med.uc.edu/proteomics • Contact • Protocols • Price list • Services • News & Events • Feedback • Publications • On-line order • FAQs

  20. FAQs • How do I get started? • What do I get for my result? • How much protein should I load for the 2-D gel analysis? • How long will it take to get the results? • Who is responsible for preparation of protein samples? • What protocol should I use for protein solubilization?

  21. Sample Preparation • The most important step towards the success of 2-D electrophoresis • Basic rules: keep everything simple use high-purity reagents • Keep proteins denatured and in solution: 7M urea, 2M thiourea, and 4% CHAPS. • Break disulfide bonds: 20 mM DTT. (NOT 2-ME) • Prevent protein modification: protease inhibitors; phosphatase inhibitors. • NO ionic detergent, esp. SDS. Non-ionic detergents are OK, e.g. Triton X-100 and NP-40. • Keep salt concentration below 10 mM. • Clean up interfering substance, including salt, nucleic acids, lipids, and polysaccharides: acetone (TCA) precipitation and commercial kits.

  22. New Fuji FLA 5100 • Pro-Q diamond fluorescent staining for phosphoproteins • Multiplexing using DIGE

  23. Multiplexing by Difference Gel Electrophoresis (DIGE) DIGE with CyDye labeling of protein (Amersham Web Site)

  24. Multiplexing by Difference Gel Electrophoresis (DIGE) • Comparison of samples within the same geleliminates gel-to-gel variation • Including the internal standards improves statistical confidence of comparing samples for protein abundance changes • Reduces number of gels needed to be run in the experiment: 2 comparing groups with 6 individuals, 36 gels for regular 2-D gels 12 gels for 2-dye DIGE 6 gels for 3-dye DIGE

  25. Post-Translational Modifications • Important for biological processes, particularly signal transductions • Most cellular processes are regulated by reversible phosphorylation of proteins • Studies of phosphorylated proteins involve radioisotope-labeling and specific antibodies

  26. Pro-Q Diamond Staining of a 2-D gel • Selectively stains phosphoproteins in 2-D gel • Allows direct in-gel detection of phosphate groups attached to tyrosine, serine, or threonine residues • NO NEED for antibody and radioisotope • Signal correlates with the number of phosphate groups • Fully compatible with mass spectrometry • Ratio of Pro-Q diamond to Sypro Ruby • =phosphorylation level/total protein

  27. Blue: Pro-Q Diamond phosphoprotein gel stain Red: SYPRO Ruby total protein stain (Molecular Probes Website)

  28. Samples Wanted… • Among 0.5~1 million proteins in human proteome, what’s your favorite one? We are here to help the hunting. • Please send your samples to: Proteomics Core MSB5301 Tel: 558-2347

  29. Special Thanks… • Dr. John Maggio • Dr. Limbach’s MS Core Dr. Stephen Macha http://www.chembus.uc.edu/massnew/maintbl.asp

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