Protein Analysis

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Protein Analysis FS Core Pat Limbach, PhD
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Protein Analysis FS Core
Objectives

• To enhance research in environmental proteomics
  by supporting proteomic services and technologies
  and
• Providing consultation and assistance with
  experimental design
• Providing consultation on data interpretation by
  taking advantage of expertise in the
  Bioinformatics Core
• Interfacing with the UC/GRI Proteomics and MS
  Facilities
• Developing and disseminating capabilities
  (Protein Production, DIGE, Protein ID, PTM
  Identification, Biomarker Discovery)

CEG
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Protein Analysis FS Core
Services

• Stimulate research of protein-environment
  interactions by providing a 50 subsidy for CEG
  members and Pilot Project awardees on
• 2-D Gel services (IEF, SDS, Staining, Imaging
  Spot Picking)
• Protein ID services (MALDI PMF, LC-MS/MS)
• Protein characterization services (PTM,
  Biomarkers)
• Bioinformatics (Modeling, Data interpretation)

CEG
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Protein Analysis FS Core
CoM 2-D Gel Services/Capabilities
Equipment/Capacity Genomic Solutions Investigator
2-D Electrophoresis System Genomic Solutions Gel
Casting System Genomic Solutions ProImage Image
Acquisition System Capacity 40 IEF strips/wk 20
SDS-PAGE gels/wk Services Complete 2-D Gel
Service (with Spot Picking for MS Analysis) IEF
Only SDS-PAGE (large format) Only Gel Staining
(fluorescent and silver staining) SDS-PAGE Plus
(includes staining imaging) Image Analysis only
or with spot picking
CEG
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Protein Analysis FS Core
GRI 2-D Gel Services/Capabilities

• Equipment/Capacity
• 4 Amersham Multiphor II IEF units for 2-D gel
  electrophoresis
• 4 Genomic Solutions Investigator units for large
  scale 2-D gels
• 5 Invitrogen XCell units for small scale 1-D and
  2-D gels
• Amersham ImageScanner imaging densitometer for
  gel scanning
• Compugen Z3 software for 2-D gel analysis
• Amersham Åkta Purifier10 HPLC system for non-gel
  protein analysis
• Services
• 1-D gel electrophoresis (small scale to medium
  scale)
• 2-D gel electrophoresis (small scale (18
  gels/wk) and large scale (40 gels/wk))
• Silver staining and Coomassie Blue staining
• Imaging densitometry of protein gels
  comparative data analysis of 2-D gels
• Western blotting

CEG
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Protein Analysis FS Core
GRI Mass Spec Services/Capabilities
Equipment Finnigan LCQ Deca XP Max with Dionex
Ultimate nanoflow 2-D HPLC Bruker Biflex III
MALDI-TOFMS Sciex API 3000 Triple quadrupole
ESI-MS with Eldex MicroPro microflow HPLC VG
Fisons Quattro Single quadrupole
ESI-MS Services MALDI Protein ID by Peptide Mass
Fingerprinting LC-MS/MS Protein ID by Sequence
Tag 2-D LC-MS/MS Protein ID of Mixtures Peptide/Me
tabolite Quantification Protein/Peptide/Oligonucle
otide Purity Confirmation
CEG
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Protein Analysis FS Core
UC Mass Spec Services/Capabilities
Equipment/Capacity Waters Q-TOF II LC-MS with
Waters Capillary HPLC Bruker Reflex IV
MALDI-TOFMS Thermo LTQ-FTMS Services MALDI
Protein ID by Peptide Mass Fingerprinting LC-MS/MS
Protein ID by Sequence Tag Accurate Mass
Measurements of Proteins Phosphorylation Analysis
by IMAC (Magnetic Bead) Purification MALDI
and/or LC-MS/MS analysis (Protocol developed
through CEG funding) Biomarker Discovery by
ClinProt Magnetic Bead Purification MALDI-MS
(Protocol developed through CEG funding)
CEG
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Protein Analysis FS Core
GRI Protein Production Services/Capabilities

• Equipment
• Biostat C-DCU
• CellTrol
• Äkta Explorer
• Services
• Subcloning of gene of interest into expression
  construct and sequencing
• Optimization of fermentation growth conditions
• Fermentation of the bacterial (12 L) or insect
  cell (4-32 L) culture containing the gene of
  interest
• Preparation of cell-free extracts
• Optimization of purification method, purification
  of the protein of interest (Quantities typically
  range from 10 mg to over 1 g depending on
  conditions)
• Personnel
• DJ Ferguson, PhD (GRI)

CEG
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Protein Analysis FS Core
Overview of Process
CEG Investigator
Protein Analysis FS Core LeaderBioinformatics
Consultant
Sample Submission
Facility Cores(Protein Production Facility)(2-D
Gel) (MS Services)
Data/Product Generation
Facility Personnel Bioinformatics Consultant
CEG Investigator
CEG
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Protein Analysis FS Core
Step 1 Information Required

• The following information will be required to
  insure the researcher obtains appropriate FS
  Core services
• Sample type? purified protein, simple mixture
  (less than 10 proteins), or complex mixture
  (e.g., cell lysates, biological fluid like serum)
• Amount of sample?
• Information desired?relative protein abundances
  or comparative analysis with no identification of
  proteins, relative protein abundances or
  comparative analysis with subsequent
  identification of proteins, protein
  identification only, or protein identification
  plus additional characterization of protein
  modification(s)

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Protein Analysis FS Core
Step 2 Initial Consultation

• Once initial information is obtained
• Contact Protein Analysis FS Core Leader to
  schedule consultation
• During consultation, appropriate facilities and
  services will be determined
• Investigator will be instructed on sample
  submission and appropriateness and mechanisms of
  CEG supplement

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Protein Analysis FS Core
Step 3 Data Review

• After services have been obtained
• Facility staff will work with investigator on
  obtaining results
• When necessary, consultation with bioinformatics
  specialists will be arranged to review data and
  findings

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Protein Analysis FS Core
Proteomics Pilot Projects/Additional Workshops

• The Protein Analysis FS Core is arranging to
  issue a request for new mini-pilot projects in
  proteomics.
• First such project funded development of protein
  profiling (Michael Borchers)
• Emphasis on projects requiring integration of
  sample preparation/data collection/data analysis
• Expectation will be one project requiring
  existing capabilities and another project
  developing new skill sets within the core
• The Protein Analysis FS Core will also be
  hosting several additional hands-on training and
  research-oriented workshops. Training/Workshop
  topics or ideas are welcome.

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Protein Analysis FS Core
Contact Information
Prof. Pat Limbach, Core Leader Pat.Limbach_at_uc.edu
556-1871 Dr. Detlef Schumann, Director GRI
Proteomics Lab schumad_at_ucmail.uc.edu
558-8950 Prof. Michael Wagner, CCHMC, Proteomics
Bioinformatics mwagner_at_cchmc.org 636-2935 Dr.
Yu Liang, Pharmacology, COM Proteomics
Core liangyu_at_ucmail.uc.edu 558-2347 Prof. Scott
Belcher, Pharmacology, 2-D Gel Consultant belches
m_at_ucmail.uc.edu 558-1721 Dr. Stephen Macha, UC
Mass Spectrometry Facility, Proteomics
Consultant machasf_at_email.uc.edu 556-3950
CEG