Bringing Proteomics to the Bringing Proteomics to the Undergraduate LaboratoryUndergraduate Laboratory
Eric S. Eberhardt and Elisa WoolridgeEric S. Eberhardt and Elisa WoolridgeDepartment of ChemistryDepartment of Chemistry
Vassar CollegeVassar CollegeDepartment of Chemistry and PhysicsDepartment of Chemistry and Physics
Marist CollegeMarist College
Lexicon of the Post-Genome Lexicon of the Post-Genome EraEra
Genomics Genomics -determine the structure and organization -determine the structure and organization of a genome as well as variations between speciesof a genome as well as variations between species
Bioinformatics Bioinformatics -extracts or mines biological -extracts or mines biological information from DNA sequence informationinformation from DNA sequence information
Functional and Structural GenomicsFunctional and Structural Genomics -shifts the -shifts the emphasis from mapping the genomes to determining emphasis from mapping the genomes to determining the biological function of open reading frames or the biological function of open reading frames or determination of three-dimensional structures of determination of three-dimensional structures of proteins proteins
What is Proteomics?What is Proteomics?
Proteome-PROTEins expressed by a Proteome-PROTEins expressed by a genOME or tissuegenOME or tissue
Proteomics Proteomics Cataloging the protein complement expressed Cataloging the protein complement expressed
by a cell or tissueby a cell or tissue Study of global changes in protein expression Study of global changes in protein expression
during development, environmental stress and during development, environmental stress and diseasedisease
Determining protein-protein interactions, Determining protein-protein interactions, yeast-two hybrid systemyeast-two hybrid system
Working Definition of Working Definition of ProteomicsProteomics
Proteomics strives to connect physiological Proteomics strives to connect physiological processes to biological pathways, regulatory processes to biological pathways, regulatory mechanisms and signaling cascades. mechanisms and signaling cascades. through the identification and quantification of proteins through the identification and quantification of proteins
expressed by a cellexpressed by a cell the localization of proteins the localization of proteins specific protein-protein interactions specific protein-protein interactions Post-translational modificationsPost-translational modifications
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
Proteomics as an Experimental Proteomics as an Experimental Approach to Biological SystemsApproach to Biological Systems
Why is Proteomics Why is Proteomics Important?Important?
Examines question not readily addressed Examines question not readily addressed by genomics or bionformaticsby genomics or bionformatics
Direct examination of gene splicing Direct examination of gene splicing productsproducts
Direct detection of post-translational Direct detection of post-translational modificationsmodifications Often associated with diseaseOften associated with disease
Pedagogical Advantages of ProteomicsPedagogical Advantages of Proteomics
Interdisciplinary area of inquiryInterdisciplinary area of inquiry Serves to capture the breadth of a student’s Serves to capture the breadth of a student’s
undergraduate experience undergraduate experience Opportunity to connect big science Opportunity to connect big science
projects, the Human Genome Project, to projects, the Human Genome Project, to laboratory experiment laboratory experiment Introduces students to both classical and Introduces students to both classical and
modern chemical and biochemical modern chemical and biochemical instrumentation and techniquesinstrumentation and techniques
Manipulation and analysis of large Manipulation and analysis of large quantities of dataquantities of data
Developments that Make Proteomics Developments that Make Proteomics Accessible to UndergraduatesAccessible to Undergraduates
Isoelectric Focusing (IEF) Cell Isoelectric Focusing (IEF) Cell Immobilized pH Gradient (IPG) Strips Immobilized pH Gradient (IPG) Strips
Fluorescent Staining and Data Analysis Fluorescent Staining and Data Analysis TechniquesTechniques SPYRO Ruby StainingSPYRO Ruby Staining 2D-Gel Electrophoresis Databases2D-Gel Electrophoresis Databases
MALDI-TOF MSMALDI-TOF MS Genomic DatabasesGenomic Databases
Overview of 2D Gel Electrophoresis
Mol
ecul
ar W
eigh
t
pH
Isoelectric Focusing (IEF)Isoelectric Focusing (IEF)
Net charge of a protein Net charge of a protein depends on pH and primary depends on pH and primary sequence of the proteinsequence of the protein
Isoelectric point (pI) is the Isoelectric point (pI) is the pH when the protein has a pH when the protein has a zero net chargezero net charge
When a protein is placed in When a protein is placed in a pH gradient and a voltage a pH gradient and a voltage is applied the protein is applied the protein migrates toward the migrates toward the cathode or anode until it cathode or anode until it reaches its pIreaches its pI
pH < pI pH = pI pH > pI
O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007
Immobilized pH Gradient (IPG) StripsImmobilized pH Gradient (IPG) Strips
CH2 CH C
O
NH2 CH2 CH C
O
NHR R = Weakly acidic or basic buffering group
Acrylamide
CH2 CH C N
O
H
CH2 N
H
C
O
CH CH2
N,N'methylenebisacrylamide
Görg, A. (2000) Electrophoresis 21, 1037 Bjellqvist, B. (1982) J. Biochem. Biophys. Methods 6, 317
IPG StripIPG Strip
CH2 CH
CO
NH
CH2 CH2 CH
CO
NH
CH
NH
CO
CHCH2
CH2 CH
CO
NH2
CH2 CH
CO
NH
CH2 CH
CO
NH2
n
n n
nCH
CH2 CH
CO
NH
CH
NH
CO
CH2 CH
CO
NH2
nCH
Crosslinked Acrylamide with ImmobilineTM
R
R
Gel Staining Gel Staining
Traditional Traditional MethodsMethods RadiolabelingRadiolabeling Sliver StainingSliver Staining
Compatibility with Compatibility with MALDI-TOF MS is MALDI-TOF MS is an issuean issue
Modern StainsModern Stains Colloidal BlueColloidal Blue
Coomassie Blue Coomassie Blue G250G250
8-50 mg protein8-50 mg protein Fluorescent StainsFluorescent Stains
SPYRO RubySPYRO Ruby Ruthenium-organic Ruthenium-organic
complexcomplex MS-Compatible MS-Compatible
Silver StainingSilver Staining ~2-4 ng protein~2-4 ng proteinColloidal Blue -Neuhoff (1988) Electrophoresis 9, 255
Ruby vs. Silver Stain -Lopez, M. F.(2000) Electrophoresis 21, 3673
SWISS-2DPAGESWISS-2DPAGETwo-dimensional Polyacrylamide Gel Two-dimensional Polyacrylamide Gel
Electrophoresis DatabaseElectrophoresis Database
Contains data on Contains data on proteins identified proteins identified and reference and reference maps of various 2-maps of various 2-D PAGE and SDS-D PAGE and SDS-PAGE gel PAGE gel
Useful for the Useful for the preliminary preliminary identification of identification of proteins by spot proteins by spot location location
http://us.expasy.org/ch2d/
Reference GelsReference GelsEE. coli. coli proteome from pH range 4.5-6.5 proteome from pH range 4.5-6.5
Proteins can Proteins can be found:be found: NameName Spot on gelSpot on gel Accession Accession
numbernumber AuthorAuthor
Spot Selection can lead to preliminary Spot Selection can lead to preliminary identification of target Proteinsidentification of target Proteins
--Heat shock protein DnaK (Hsp70)Heat shock protein DnaK (Hsp70)
MALDI-TOF Mass MALDI-TOF Mass SpectrometrySpectrometry
Sample Desorption and Sample Desorption and IonizationIonization
Time-of-FlightTime-of-Flight
zKEm
st)2(
Module Design-Six WeeksModule Design-Six Weeks
Week 1: Cell culturing and Sample Week 1: Cell culturing and Sample PreparationPreparation
Week 2: Protein Quantitation and 1Week 2: Protein Quantitation and 1stst DimensionDimension
Week 3: 2Week 3: 2ndnd Dimension and Staining Dimension and Staining
Proteomics Module OutlineProteomics Module Outline
Week 4: Spot Excision and Trypsin Week 4: Spot Excision and Trypsin DigestionDigestion
Week 5: MALDI-TOF MS AnalysisWeek 5: MALDI-TOF MS Analysis
Week 6: Database MiningWeek 6: Database Mining
Experimental OutlineExperimental Outline
E. coli E. coli K-12 MG1655 subjected to heat shock at K-12 MG1655 subjected to heat shock at 46ºC for 40 and 70 minutes46ºC for 40 and 70 minutes
Lysed-cells separated in two dimensions by Lysed-cells separated in two dimensions by isoelectric point and by massisoelectric point and by mass
Gels imaged and quantified with PDQuest Gels imaged and quantified with PDQuest SoftwareSoftware
Proteins spots excised, digested with Trypsin, and Proteins spots excised, digested with Trypsin, and subjected to MALDI-TOF MS analysissubjected to MALDI-TOF MS analysis
Protein identity established through Protein identity established through Bioinformatics using SWISS-2DPAGE and Protein Bioinformatics using SWISS-2DPAGE and Protein Prospector databasesProspector databases
Module 1: Heat Shock Module 1: Heat Shock ResponseResponse
During heat shock response-the transcription ofDuring heat shock response-the transcription of
~20 heat shock genes is initiated ~20 heat shock genes is initiated Primary protein products of heat shock genes are Primary protein products of heat shock genes are
molecular chaperones such as GroEL and GroESmolecular chaperones such as GroEL and GroES Chaperones that enhance the efficiency and recycle proteins Chaperones that enhance the efficiency and recycle proteins
in the cellin the cell Serve to break up protein aggregates, and facilitate the Serve to break up protein aggregates, and facilitate the
subsequent folding of these polypeptidessubsequent folding of these polypeptides
Molecular Chaperone GroEL/ES Molecular Chaperone GroEL/ES ComplexComplex
14 subunits each 547 14 subunits each 547 aaaa
7 subunits to each 7 subunits to each ringring
GroES subunits rest on GroES subunits rest on top to seal substrate top to seal substrate binding pocketbinding pocket
Xu, Z; Horwich, A. L., Sigler, P.B. (1997) Nature 388, p. 741Protein Data Bank (AON1)
Chaperone mediated Chaperone mediated control of peptide refoldingcontrol of peptide refolding
GroEL/GroES complex GroEL/GroES complex associates with the associates with the polypeptidepolypeptide
ADP and GroES ADP and GroES dissociate from dissociate from complexcomplex
ATP and GroES ATP and GroES associate to reform associate to reform the complexthe complex
ATP is hydrolyzedATP is hydrolyzed GroEL/GroES complex GroEL/GroES complex
disassociatesdisassociates
Polypeptide GroEL GroES
ATP
GroES
GroES
ADP
ADP
Polypeptide
Polypeptide
GroEL
GroEL GroES
ATP
GroEL GroES
ADP
Control Gel pH 4.7-5.9
40 Minute Heat Shock Gel pH 4.7-5.9
70 Minute Heat Shock Gel pH 4.7-5.9
E. coli Heat Shock 2D Gels over pH range 4.7-5.9
DnaKS1
GroELGroEL
GroES
DnaK
GroEL
S1
GroES
DnaK S1
GroEL
GroES
DnaK
DnaK-PO4
S1
GroELGroEL-PO4
Control Gel 4.7-5.9
70 Minute Heat Shock Gel 4.7-5.9
Zoomed Images of E. coli Heat Shock 2D Gels over pH range
4.7-5.9
DnaK
DnaK-PO4
S1
GroEL GroEL-PO4
DnaK
GroEL
DnaK-PO4
S1
GroEL-PO4
40 Minute Heat Shock Gel 4.7-5.9
499.0 999.4 1499.8 2000.2 2500.6 3001.0
Mass (m/z)
0
3.0E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Voyager Spec #1[BP = 2273.3, 29967]
2273.51
2163.17
659.79
1598.36
2289.24
1485.43568.73 1180.61805.75582.78 2441.25
2217.07 2653.06 2869.052224.99 2424.23
MALDI-TOF Peptide Spectrum of DnaK
1 11 21 31 41 51 61 71
GKIIGIDLGT TNSCVAIMDG TTPRVLENAE GDRTTPSIIA YTQDGETLVG QPAKRQAVTN PQNTLFAIKR LIGRRFQDEE
81 91 101 111 121 131 141 151
VQRDVSIMPF KIIAADNGDA WVEVKGQKMA PPQISAEVLK KMKKTAEDYL GEPVTEAVIT VPAYFNDAQR QATKDAGRIA
161 171 181 191 201 211 221 231
GLEVKRIINE PTAAALAYGL DKGTGNRTIA VYDLGGGTFD ISIIEIDEVD GEKTFEVLAT NGDTHLGGED FDSRLINYLV
241 251 261 271 281 291 301 311
EEFKKDQGID LRNDPLAMQR LKEAAEKAKI ELSSAQQTDV NLPYITADAT GPKHMNIKVT RAKLESLVED LVNRSIEPLK
321 331 341 351 361 371 381 391
VALQDAGLSV SDIDDVILVG GQTRMPMVQK KVAEFFGKEP RKDVNPDEAV AIGAAVQGGV LTGDVKDVLL LDVTPLSLGI
401 411 421 431 441 451 461 471
ETMGGVMTTL IAKNTTIPTK HSQVFSTAED NQSAVTIHVL QGERKRAADN KSLGQFNLDG INPAPRGMPQ IEVTFDIDAD
481 491 501 511 521 531 541 551
GILHVSAKDK NSGKEQKITI KASSGLNEDE IQKMVRDAEA NAEADRKFEE LVQTRNQGDH LLHSTRKQVE EAGDKLPADD
561 571 581 591 601 611 621 631
KTAIESALTA LETALKGEDK AAIEAKMQEL AQVSQKLMEI AQQQHAQQQT AGADASANNA KDDDVVDAEF EEVKDKK
m/z submitted MH+ matched Δ ppm Fragment Start (AA #) Fragment End (AA#) Peptide Sequence1050.5412 1050.4856 52.9497 76 83 (R) FQDEEVQR(D) 1179.6231 1179.6162 5.8168 352 361 (K) VAEFFGKEPR(K) 2346.0767 2346.2305 -65.5554 34 55 (R) TTPSIIAYTQDGETLVGQPAKR(Q) 2441.2316 2441.2888 -23.4185 321 344 (K) VALQDAGLSVSDIDDVILVGGQTR(M) 2653.2858 2653.297 -4.233 421 444 (K) HSQVFSTAEDNQSAVTIHVLQGER(K)
Peptide fingerprint of DnaK including matched peaks and their corresponding sequences determined through MALDI analysis
Quantification of Heat Shock Protein Expression Over Time
ProteinProtein Mass Mass (kD)(kD)
pIpI MOWSE MOWSE scorescore
Fold Fold IncreaseIncrease
DnaKDnaK 68.9868.98 4.834.83 2740027400 1.51.5
GroELGroEL 57.1457.14 4.854.85 40004000 1.91.9
GroESGroES 10.3910.39 5.155.15 1460014600 2020
30S R-30S R-subunitsubunit
61.1661.16 4.894.89 912912 3.63.6
Module Variation: Heat Shock Module Variation: Heat Shock vs. Gradual Temperature vs. Gradual Temperature
IncreaseIncrease Student Designed experimentStudent Designed experiment Are Hsp Expression levels the same for a 16 Are Hsp Expression levels the same for a 16 °°C jump C jump
vs 16 vs 16 °°C gradual increase in temperature?C gradual increase in temperature? Jump Conditions:Jump Conditions:
Growth to ODGrowth to OD595595 = 0.4 at 30 = 0.4 at 30 °°C then warm to 46 C then warm to 46 °°C in 5 minC in 5 min
Gradual Increase: Gradual Increase: Growth to ODGrowth to OD595595 = 0.4 at 30 = 0.4 at 30 °°C then warm to 46 C then warm to 46 °°C over 60 C over 60
minmin
Use Swiss 2d Gel Database to determine protein Use Swiss 2d Gel Database to determine protein identityidentity
Major Hsp region Major Hsp region Control at 30 Control at 30 °°CC
t = 0 t = 30
t = 60 t = 90
Major Hsp region Major Hsp region Gradual Increase to 46 Gradual Increase to 46 °°C (1 C (1
hr)hr) t = 0 t = 30 t = 60
t = 90 t = 120
Major Hsp Region Major Hsp Region Jump ExperimentJump Experiment
Last Time Point of Gradual Expt
t = 0 t = 30 min
t = 60 min
t = 120
Module 2: Cold Shock Module 2: Cold Shock AdaptationAdaptation
Family of Csp’s involved in stabilizing translational Family of Csp’s involved in stabilizing translational machinery and alter membrane fluiditymachinery and alter membrane fluidity
Response is induced by transient blockage of Response is induced by transient blockage of translation initiationtranslation initiation
13 Polypeptides are induced13 Polypeptides are induced 10-fold induction observed for many csp10-fold induction observed for many csp
Other induced proteins include: CspB, CspG, RecA, DNA Other induced proteins include: CspB, CspG, RecA, DNA gyrase, NusAgyrase, NusA
Module 2: Cold Shock Module 2: Cold Shock AdaptationAdaptation
Csps are fairly small ~7 KDCsps are fairly small ~7 KD
CspA and CspB have CspA and CspB have similar tertiary structuressimilar tertiary structures
Binds single stranded RNABinds single stranded RNA
CspA binds mRNA and acts CspA binds mRNA and acts as an mRNA chaperoneas an mRNA chaperone
Schindelin, H. Proc Natl Acad Sci U S A 91 pp. 5119 (1994)
Cold Shock ResponseCold Shock Response
Control gel 37 ºC
Cold Shock at 16 Cold Shock at 16 ººCC
Cold Shock SummaryCold Shock Summary
Significant differences Significant differences are observed in the are observed in the proteomeproteome
Observe induction of Observe induction of CspA, CspD and CspGCspA, CspD and CspG
Transient increase in Transient increase in other Stress related other Stress related proteins including proteins including DnaK and GroELDnaK and GroEL
Other Planned Environmental Other Planned Environmental Stress ModulesStress Modules
Oxidative StressOxidative Stress Osmotic StressOsmotic Stress pH StresspH Stress AntibioticAntibiotic Recombinant Protein ExpressionRecombinant Protein Expression RemediationRemediation
Module 3: New Approach to Teaching MetabolismModule 3: New Approach to Teaching MetabolismGrowth on Different Carbon SourcesGrowth on Different Carbon Sources
Glucose vs. Acetate Glucose vs. Acetate
37 37 °°C Minimal Media C Minimal Media GlucoseGlucose
pH 4-7
37 37 °°C Minimal Media C Minimal Media AcetateAcetate
pH 4-7
Carbon Source SummaryCarbon Source Summary
Clear differences between the two growth conditions
Glucose media Acetate Media
Evaluation PlanEvaluation Plan
NSF CCLI-EMD “Proof-of-concept” Program NSF CCLI-EMD “Proof-of-concept” Program GoalsGoals
“…“…develop materials that incorporate develop materials that incorporate effective educational practices…”effective educational practices…”
““A pilot test that provides a credible A pilot test that provides a credible evaluation of the prototype”evaluation of the prototype”
Three Phase Plan Three Phase Plan -Two External Consultants-Two External Consultants
Education Evaluators- design Education Evaluators- design evaluation plan and provide a report evaluation plan and provide a report on the effectiveness of the projecton the effectiveness of the project
Biochemist- to evaluate the materials Biochemist- to evaluate the materials and scientific merit of the proteomic and scientific merit of the proteomic modulesmodules
Quantitative Evaluation of Quantitative Evaluation of Educational EffectivenessEducational Effectiveness
Pre-test and Post-test Week 1, Week 3 and Pre-test and Post-test Week 1, Week 3 and Week 6Week 6 Designed to evaluate the increase in student Designed to evaluate the increase in student
understanding of basis of specific techniques and understanding of basis of specific techniques and details of the biological system they are studyingdetails of the biological system they are studying
Conducted On-line in a multiple choice formatConducted On-line in a multiple choice format
On-line evaluation of student On-line evaluation of student response/satisfactionresponse/satisfaction
Evaluation of Critical Thinking Evaluation of Critical Thinking SkillsSkills
Short Research Proposals Short Research Proposals Design an experiment to determine the Design an experiment to determine the
regulatory proteins of an environmental regulatory proteins of an environmental stressstress
Pre-Test and Post-testPre-Test and Post-test
Evaluated by InstructorEvaluated by Instructor
Biochemical ContentBiochemical Content
External Evaluation of Course ManualExternal Evaluation of Course Manual
Review of Student Laboratory Review of Student Laboratory Reports Reports
Review of Videotape Student Oral Review of Videotape Student Oral PresentationsPresentations
AcknowledgementsAcknowledgements
NSF-CCLI ProgramNSF-CCLI Program NSF-MRI ProgramNSF-MRI Program HHMI University AwardHHMI University Award
Vassar Biochemistry Vassar Biochemistry Seniors from the Classes Seniors from the Classes of 2001 and 2002of 2001 and 2002
Brett Spain Marist Brett Spain Marist CollegeCollege
