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Bringing Proteomics to the Undergraduate Laboratory

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Bringing Proteomics to the Undergraduate Laboratory Eric S. Eberhardt and Elisa Woolridge Department of Chemistry Vassar College Department of Chemistry and Physics – PowerPoint PPT presentation

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Title: 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 students
    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 lt pI pH pI pH gt pI
OFarrell, 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
Gel Staining
  • 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

Colloidal Blue -Neuhoff (1988) Electrophoresis 9,
255 Ruby vs. Silver Stain -Lopez, M. F.(2000)
Electrophoresis 21, 3673
14
SWISS-2DPAGE Two-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 Gels E. 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
S1
DnaK-PO4
DnaK-PO4
S1
DnaK
DnaK
GroEL-PO4
GroEL
GroEL
GroEL-PO4
70 Minute Heat Shock Gel 4.7-5.9
40 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
Protein Mass (kD) pI MOWSE score Fold Increase
DnaK 68.98 4.83 27400 1.5
GroEL 57.14 4.85 4000 1.9
GroES 10.39 5.15 14600 20
30S R-subunit 61.16 4.89 912 3.6
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 Csps 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
Metabolism Growth 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/satisfactio
    n

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
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