Bringing Proteomics to the Undergraduate Laboratory

1. Bringing Proteomics to the Undergraduate Laboratory Eric S. Eberhardt and Elisa Woolridge Department of Chemistry Vassar College Department of Chemistry and Physics Marist College

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. Overview of 2D Gel Electrophoresis Molecular Weight pH

10. 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

11. Immobilized pH Gradient (IPG) Strips Görg, A. (2000) Electrophoresis 21, 1037 Bjellqvist, B. (1982) J. Biochem. Biophys. Methods 6, 317

12. IPG Strip

13. 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

14. 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/

15. Reference GelsE. coli proteome from pH range 4.5-6.5 • Proteins can be found: • Name • Spot on gel • Accession number • Author

16. Spot Selection can lead to preliminary identification of target Proteins-Heat shock protein DnaK (Hsp70)

17. MALDI-TOF Mass Spectrometry

18. Sample Desorption and Ionization

19. Time-of-Flight

20. Module Design-Six Weeks • Week 1: Cell culturing and Sample Preparation • Week 2: Protein Quantitation and 1st Dimension • Week 3: 2nd Dimension and Staining

21. Proteomics Module Outline • Week 4: Spot Excision and Trypsin Digestion • Week 5: MALDI-TOF MS Analysis • Week 6: Database Mining

22. 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

23. 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

24. 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)

25. 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

26. 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

27. 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

28. MALDI-TOF Peptide Spectrum of DnaK

29. Peptide fingerprint of DnaK including matched peaks and their corresponding sequences determined through MALDI analysis

30. Quantification of Heat Shock Protein Expression Over Time

31. 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

32. Major Hsp region Control at 30 °C t = 0 t = 30 t = 60 t = 90

33. Major Hsp region Gradual Increase to 46 °C (1 hr) t = 0 t = 30 t = 60 t = 90 t = 120

34. Major Hsp Region Jump Experiment t = 0 t = 30 min t = 120 t = 60 min Last Time Point of Gradual Expt

35. 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

36. 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)

37. Cold Shock Response Control gel 37 ºC

38. Cold Shock at 16 ºC

39. 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

40. Other Planned Environmental Stress Modules • Oxidative Stress • Osmotic Stress • pH Stress • Antibiotic • Recombinant Protein Expression • Remediation

41. Module 3: New Approach to Teaching MetabolismGrowth on Different Carbon Sources Glucose vs. Acetate

42. 37 °C Minimal Media Glucose pH 4-7

43. 37 °C Minimal Media Acetate pH 4-7

44. Carbon Source Summary Glucose media Acetate Media Clear differences between the two growth conditions

45. 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”

46. 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

47. 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

48. 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

49. Biochemical Content • External Evaluation of Course Manual • Review of Student Laboratory Reports • Review of Videotape Student Oral Presentations

50. Acknowledgements • NSF-CCLI Program • NSF-MRI Program • HHMI University Award • Vassar Biochemistry Seniors from the Classes of 2001 and 2002 • Brett Spain Marist College