500 likes | 634 Views
Bringing Proteomics to the Undergraduate Laboratory. Eric S. Eberhardt and Elisa Woolridge Department of Chemistry Vassar College Department of Chemistry and Physics Marist College. Lexicon of the Post-Genome Era.
E N D
Bringing Proteomics to the Undergraduate Laboratory Eric S. Eberhardt and Elisa Woolridge Department of Chemistry Vassar College Department of Chemistry and Physics Marist College
Lexicon of the Post-Genome Era • Genomics -determine the structure and organization of a genome as well as variations between species • Bioinformatics -extracts or mines biological information from DNA sequence information • Functional and Structural Genomics -shifts the emphasis from mapping the genomes to determining the biological function of open reading frames or determination of three-dimensional structures of proteins
What is Proteomics? • Proteome-PROTEins expressed by a genOME or tissue • Proteomics • Cataloging the protein complement expressed by a cell or tissue • Study of global changes in protein expression during development, environmental stress and disease • Determining protein-protein interactions, yeast-two hybrid system
Working Definition of Proteomics • Proteomics strives to connect physiological processes to biological pathways, regulatory mechanisms and signaling cascades. • through the identification and quantification of proteins expressed by a cell • the localization of proteins • specific protein-protein interactions • Post-translational modifications
Proteomics as an Experimental Approach to Biological Systems pI Size 2-D gel electrophoresis of sample Excise spot, destain, digest with bovine trypsin Extract peptides and analyze with MALDI-TOF MS Database mining
Why is Proteomics Important? • Examines question not readily addressed by genomics or bionformatics • Direct examination of gene splicing products • Direct detection of post-translational modifications • Often associated with disease
Pedagogical Advantages of Proteomics • Interdisciplinary area of inquiry • Serves to capture the breadth of a student’s undergraduate experience • Opportunity to connect big science projects, the Human Genome Project, to laboratory experiment • Introduces students to both classical and modern chemical and biochemical instrumentation and techniques • Manipulation and analysis of large quantities of data
Developments that Make Proteomics Accessible to Undergraduates • Isoelectric Focusing (IEF) Cell • Immobilized pH Gradient (IPG) Strips • Fluorescent Staining and Data Analysis Techniques • SPYRO Ruby Staining • 2D-Gel Electrophoresis Databases • MALDI-TOF MS • Genomic Databases
Overview of 2D Gel Electrophoresis Molecular Weight pH
Isoelectric Focusing (IEF) • Net charge of a protein depends on pH and primary sequence of the protein • Isoelectric point (pI) is the pH when the protein has a zero net charge • When a protein is placed in a pH gradient and a voltage is applied the protein migrates toward the cathode or anode until it reaches its pI pH < pI pH = pI pH > pI O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007
Immobilized pH Gradient (IPG) Strips Görg, A. (2000) Electrophoresis 21, 1037 Bjellqvist, B. (1982) J. Biochem. Biophys. Methods 6, 317
Traditional Methods Radiolabeling Sliver Staining Compatibility with MALDI-TOF MS is an issue Modern Stains Colloidal Blue Coomassie Blue G250 8-50 mg protein Fluorescent Stains SPYRO Ruby Ruthenium-organic complex MS-Compatible Silver Staining ~2-4 ng protein Gel Staining Colloidal Blue -Neuhoff (1988) Electrophoresis 9, 255 Ruby vs. Silver Stain -Lopez, M. F.(2000) Electrophoresis 21, 3673
SWISS-2DPAGETwo-dimensional Polyacrylamide Gel Electrophoresis Database • Contains data on proteins identified and reference maps of various 2-D PAGE and SDS-PAGE gel • Useful for the preliminary identification of proteins by spot location http://us.expasy.org/ch2d/
Reference GelsE. coli proteome from pH range 4.5-6.5 • Proteins can be found: • Name • Spot on gel • Accession number • Author
Spot Selection can lead to preliminary identification of target Proteins-Heat shock protein DnaK (Hsp70)
Module Design-Six Weeks • Week 1: Cell culturing and Sample Preparation • Week 2: Protein Quantitation and 1st Dimension • Week 3: 2nd Dimension and Staining
Proteomics Module Outline • Week 4: Spot Excision and Trypsin Digestion • Week 5: MALDI-TOF MS Analysis • Week 6: Database Mining
Experimental Outline • E. coli K-12 MG1655 subjected to heat shock at 46ºC for 40 and 70 minutes • Lysed-cells separated in two dimensions by isoelectric point and by mass • Gels imaged and quantified with PDQuest Software • Proteins spots excised, digested with Trypsin, and subjected to MALDI-TOF MS analysis • Protein identity established through Bioinformatics using SWISS-2DPAGE and Protein Prospector databases
Module 1: Heat Shock Response • During heat shock response-the transcription of ~20 heat shock genes is initiated • Primary protein products of heat shock genes are molecular chaperones such as GroEL and GroES • Chaperones that enhance the efficiency and recycle proteins in the cell • Serve to break up protein aggregates, and facilitate the subsequent folding of these polypeptides
Molecular Chaperone GroEL/ES Complex • 14 subunits each 547 aa • 7 subunits to each ring • GroES subunits rest on top to seal substrate binding pocket Xu, Z; Horwich, A. L., Sigler, P.B. (1997) Nature 388, p. 741 Protein Data Bank (AON1)
Chaperone mediated control of peptide refolding GroEL Polypeptide ADP GroES ADP GroES • GroEL/GroES complex associates with the polypeptide • ADP and GroES dissociate from complex • ATP and GroES associate to reform the complex • ATP is hydrolyzed • GroEL/GroES complex disassociates GroEL Polypeptide ATP GroES GroEL ATP Polypeptide GroES GroEL ADP GroES
DnaK E. coli Heat Shock 2D Gels over pH range 4.7-5.9 S1 GroEL GroEL GroES Control Gel pH 4.7-5.9 DnaK S1 GroEL GroES 40 Minute Heat Shock Gel pH 4.7-5.9 DnaK S1 GroEL GroES 70 Minute Heat Shock Gel pH 4.7-5.9
DnaK-PO4 S1 Zoomed Images of E. coli Heat Shock 2D Gels over pH range 4.7-5.9 DnaK GroEL GroEL-PO4 Control Gel 4.7-5.9 DnaK-PO4 S1 DnaK-PO4 S1 DnaK DnaK GroEL-PO4 GroEL GroEL GroEL-PO4 40 Minute Heat Shock Gel 4.7-5.9 70 Minute Heat Shock Gel 4.7-5.9
Peptide fingerprint of DnaK including matched peaks and their corresponding sequences determined through MALDI analysis
Module Variation: Heat Shock vs. Gradual Temperature Increase • Student Designed experiment • Are Hsp Expression levels the same for a 16 °C jump vs 16 °C gradual increase in temperature? • Jump Conditions: • Growth to OD595 = 0.4 at 30 °C then warm to 46 °C in 5 min • Gradual Increase: • Growth to OD595 = 0.4 at 30 °C then warm to 46 °C over 60 min • Use Swiss 2d Gel Database to determine protein identity
Major Hsp region Control at 30 °C t = 0 t = 30 t = 60 t = 90
Major Hsp region Gradual Increase to 46 °C (1 hr) t = 0 t = 30 t = 60 t = 90 t = 120
Major Hsp Region Jump Experiment t = 0 t = 30 min t = 120 t = 60 min Last Time Point of Gradual Expt
Module 2: Cold Shock Adaptation • Family of Csp’s involved in stabilizing translational machinery and alter membrane fluidity • Response is induced by transient blockage of translation initiation • 13 Polypeptides are induced • 10-fold induction observed for many csp • Other induced proteins include: CspB, CspG, RecA, DNA gyrase, NusA
Module 2: Cold Shock Adaptation • Csps are fairly small ~7 KD • CspA and CspB have similar tertiary structures • Binds single stranded RNA • CspA binds mRNA and acts as an mRNA chaperone Schindelin, H. Proc Natl Acad Sci U S A 91 pp. 5119 (1994)
Cold Shock Response Control gel 37 ºC
Cold Shock Summary • Significant differences are observed in the proteome • Observe induction of CspA, CspD and CspG • Transient increase in other Stress related proteins including DnaK and GroEL
Other Planned Environmental Stress Modules • Oxidative Stress • Osmotic Stress • pH Stress • Antibiotic • Recombinant Protein Expression • Remediation
Module 3: New Approach to Teaching MetabolismGrowth on Different Carbon Sources Glucose vs. Acetate
37 °C Minimal Media Glucose pH 4-7
37 °C Minimal Media Acetate pH 4-7
Carbon Source Summary Glucose media Acetate Media Clear differences between the two growth conditions
Evaluation Plan • NSF CCLI-EMD “Proof-of-concept” Program Goals • “…develop materials that incorporate effective educational practices…” • “A pilot test that provides a credible evaluation of the prototype”
Three Phase Plan -Two External Consultants • Education Evaluators- design evaluation plan and provide a report on the effectiveness of the project • Biochemist- to evaluate the materials and scientific merit of the proteomic modules
Quantitative Evaluation of Educational Effectiveness • Pre-test and Post-test Week 1, Week 3 and Week 6 • Designed to evaluate the increase in student understanding of basis of specific techniques and details of the biological system they are studying • Conducted On-line in a multiple choice format • On-line evaluation of student response/satisfaction
Evaluation of Critical Thinking Skills • Short Research Proposals • Design an experiment to determine the regulatory proteins of an environmental stress • Pre-Test and Post-test • Evaluated by Instructor
Biochemical Content • External Evaluation of Course Manual • Review of Student Laboratory Reports • Review of Videotape Student Oral Presentations
Acknowledgements • NSF-CCLI Program • NSF-MRI Program • HHMI University Award • Vassar Biochemistry Seniors from the Classes of 2001 and 2002 • Brett Spain Marist College