CEG Protein Analysis WorkshopCEG Protein Analysis Workshop

Two-Dimensional Gel Electrophoresis

Yu Liang, Ph.D.

Proteomics Core Facility at UC Medical Center

From Genotype to PhenotypeFrom Genotype to Phenotype

Genome: DNAsTranscriptome: RNAsProteome: ProteinsPhysiome: MetabolitesBiome: Environment

Beyond the GenomeBeyond 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)

Proteomics OverviewProteomics Overview

Sample Preparation 2-D Electrophoresis Spot Detection & Image Analysis

Enzymatic Digestion Peptide-Mass Fingerprinting

Peptide Sequencing via MS Database Search

Protein Identification

(I) (III)

(IV) (V) (VI)

(II)

PersonnelPersonnel

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

Customized Core ServicesCustomized 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

Proteomics Core EquipmentProteomics 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)

Genomic Solutions 2D Electrophoresis Genomic Solutions 2D Electrophoresis SystemSystem

pH phaser isoelectric focusing system

Investigator 2-D electrophoresisrunning system

2-D Image (Sypro Ruby)2-D Image (Sypro Ruby)

• mouse cardiac

• 250 g loading

• pH 3-10 IEF strips

• 12.5% SDS-PAGE

• File ID: mb699

pH lowMW

High

High

low

Sample gelsSample gels

Control Experimental

2-D Image2-D Image

Control

• mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso; spot ID: S8-1

Experimental

2-D Image 2-D Image

• mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso

Control Experimental

2-D Image2-D Image

• mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc5bcon vs. sc15iso

Control

Experimental

PTM?

Downregulation?

2-D Gel Analysis2-D Gel Analysis

2-D Gel Analysis2-D Gel Analysis

2-D Gel Analysis2-D Gel Analysis

DeliverablesDeliverables

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

New WebsiteNew Website

Contact Protocols Price list Services News & Events Feedback Publications On-line order FAQs

www.med.uc.edu/proteomics or proteomics.uc.eduor proteomics.uc.edu

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?

FAQsFAQs

Sample PreparationSample Preparation

The most important step towards the success of 2-D electrophoresis Basic rules: keep everything simple keep samples cool 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. Protein quantity measurement

New Fuji FLA 5100New Fuji FLA 5100

Pro-Q diamond fluorescent staining for phosphoproteins

Multiplexing using DIGE

Multiplexing by Difference Gel Electrophoresis Multiplexing by Difference Gel Electrophoresis (DIGE)(DIGE)

DIGE with CyDye labeling of protein

(Amersham Web Site)

Multiplexing by Difference Gel Electrophoresis Multiplexing by Difference Gel Electrophoresis (DIGE)(DIGE)

Comparison of samples within the same gel eliminates 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

Post-Translational ModificationsPost-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

Pro-Q Diamond Staining of a 2-D gelPro-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

Pro-Q Diamond staining Pro-Q Diamond staining

Blue: Pro-Q Diamond phosphoprotein gel stain Red: SYPRO Ruby total protein stain

(Molecular Probes Website)

Samples Wanted…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: (513) 558-2347

Current/Prospective ClientsCurrent/Prospective Clients

Simon Chu – Pharmacology Yolanda Sanchez – Mol. Genetics Keith Jones – Pharmacology Kathleen Dixon – Env. Health Leslie Myatt – OB/GYN Ron Millard – Pharmacology Alex Lentsch – Surgery William Martin – Dean’s Lab/Administrative Evangelia Kranias – Pharmacology Jeff Molkentin – CHMC/Mol. Card. Biology Jeff Robbins – CHMC/Mol. Card. Biology Jennifer Maller – CHMC/Plastic Surgery Alvaro Puga – Environmental Health Howard Shertzer – Envoironmental Health Frank Sharp – Neurology Erik Knudsen – Cell Biology Karen Knudsen – Cell Biology Raymond Boissy – Dermatology

Jodie Duffy – CHMC/Cardiovascular Surgery Kristen Page – CHMC/Critical Care Joanna Groden – Molecular Genetics David Wieczorek – Molecular Genetics Joan Cook-Mills – Pathology Muhammed Ashraf – Pathology Jim Stringer – Molecular Genetics Ralph Giannella – IM/Digestive Disease Steven Keller – Biology Joseph Solomkin – Surgery Tom Shanley – CHMC/Critical Care Steven Chernausek – CHMC/Endocrinology George Leikauf – Environmental Health Michael Borchers – Environmental Health Gary Shull – Molecular Genetics Jeff Matthews – Surgery Mary-Beth Genter – Environmental Health

Special Thanks…Special Thanks…

Dr. John Maggio

Dr. Limbach’s MS CoreDr. Stephen Macha

http://www.chembus.uc.edu/massnew/maintbl.asp