CEG Protein Analysis F&S Core Pat Limbach, PhD

CEG 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) Protein Analysis F&S Core

CEG 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) Services Protein Analysis F&S Core

CEG 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 CoM 2-D Gel Services/Capabilities Protein Analysis F&S Core

CEG 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 GRI 2-D Gel Services/Capabilities Protein Analysis F&S Core

CEG 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 GRI Mass Spec Services/Capabilities Protein Analysis F&S Core

CEG 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) UC Mass Spec Services/Capabilities Protein Analysis F&S Core

CEG 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) GRI Protein Production Services/Capabilities Protein Analysis F&S Core

CEG Overview of Process Protein Analysis F&S Core CEG InvestigatorProtein Analysis F&S Core Leader Bioinformatics Consultant Facility Cores (Protein Production Facility) (2-D Gel) (MS Services) Facility Personnel Bioinformatics Consultant CEG Investigator Sample Submission Data/Product Generation

CEG Step 1: Information Required Protein Analysis F&S Core 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 Step 2: Initial Consultation Protein Analysis F&S Core 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 Step 3: Data Review Protein Analysis F&S Core 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 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. Proteomics Pilot Projects/Additional Workshops Protein Analysis F&S Core

CEG Prof. Pat Limbach, Core Leader Dr. Detlef Schumann, Director GRI Proteomics Lab Prof. Michael Wagner, CCHMC, Proteomics Bioinformatics Dr. Yu Liang, Pharmacology, COM Proteomics Core Prof. Scott Belcher, Pharmacology, 2-D Gel Consultant Dr. Stephen Macha, UC Mass Spectrometry Facility, Proteomics Consultant Contact Information Protein Analysis F&S Core