Protein Analysis
F&S Core
Pat Limbach, PhD
CEG
Protein Analysis F&S 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
Protein Analysis F&S 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
Protein Analysis F&S 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
Protein Analysis F&S 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
Protein Analysis F&S 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/Metabolite Quantification
Protein/Peptide/Oligonucleotide Purity Confirmation
CEG
Protein Analysis F&S 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
Protein Analysis F&S 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
Protein Analysis F&S Core
Overview of Process
CEG Investigator
Protein Analysis F&S Core Leader
Bioinformatics Consultant
Sample Submission
Facility Cores
(Protein Production Facility)
(2-D Gel)
(MS Services)
Data/Product Generation
CEG Investigator
Facility Personnel
Bioinformatics Consultant
CEG
Protein Analysis F&S Core
Step 1: Information Required
The following information will be required to insure the researcher
obtains appropriate F&S Core services:
1. Sample type?
purified protein, simple mixture (less than 10 proteins), or complex mixture
(e.g., cell lysates, biological fluid like serum)
2. Amount of sample?
3. 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)
CEG
Protein Analysis F&S Core
Step 2: Initial Consultation
Once initial information is obtained:
• Contact Protein Analysis F&S 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
CEG
Protein Analysis F&S 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
CEG
Protein Analysis F&S Core
Proteomics Pilot Projects/Additional Workshops
The Protein Analysis F&S 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 F&S Core will also be hosting several
additional hands-on training and research-oriented workshops.
Training/Workshop topics or ideas are welcome.
CEG
Protein Analysis F&S Core
Contact Information
Prof. Pat Limbach, Core Leader
Pat.Limbach@uc.edu; 556-1871
Dr. Detlef Schumann, Director GRI Proteomics Lab
schumad@ucmail.uc.edu; 558-8950
Prof. Michael Wagner, CCHMC, Proteomics Bioinformatics
mwagner@cchmc.org; 636-2935
Dr. Yu Liang, Pharmacology, COM Proteomics Core
liangyu@ucmail.uc.edu; 558-2347
Prof. Scott Belcher, Pharmacology, 2-D Gel Consultant
belchesm@ucmail.uc.edu; 558-1721
Dr. Stephen Macha, UC Mass Spectrometry Facility, Proteomics Consultant
machasf@email.uc.edu; 556-3950
CEG