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Transcript
I MAGE G ALLERY Probing 100 fmole of digested HSA by Nano LC-LTQ/MS/MS P ROTEOMICS F ACILITY S CIENTIFIC O BJECTIVES Proteomics Facility at the University of Vermont is a core facility funded by the Vermont Genetics Network to serve the Lead Institution and research throughout the state as part of the IDeA Program mission of building research infrastructure. The facility provides a central resource of mass spectrometry-based proteomics technologies to identify, characterize and quantify target proteins in various biological and biomedical samples. We strive to establish a highly efficient research and educational environment for sharing ideas, experiences, and knowledge of proteomics application in systems biology, biomedical and clinical studies. UVM/VGN PROTEOMICS FACILITY High Quality for Low Rates! Determination of Phosphorylation Sites in GSK3-beta after Drug Treatment Consultation Free Coomassie Blue-stained gel band $10/band Silver-stained gel band $25/band Data analysis (bioinformatics) Free Method development Free Marsh Life Science 335 F ACILITY Number of sample submissions I NFORMATION Number of facility users VGN Proteomics University of Vermont Marsh Life Science 335 Burlington,VT 05405 Identification of Pafa1b2 from Human Testis Homogenate http://vgn.uvm.edu/proteomics * * Quantification of Protein Expression in Biomedical Samples More information about our facility can be found on our website (http://vgn.uvm.edu/proteomics) VGN is funded by a grant from the National Institute of Health F ACILITY I NFORMATION Services Provided: Protein identification, isoform characterization and molecular weight determination Identification of posttranslational modifications (PTMs; e.g. phosphorylation, N-glycosylation, methylation, acetylation, ubiquitination, tyrosine nitration, S-nitrosylation, etc) Identification of protein-protein interacting partners and determination of their binding stoichiometries De novo sequencing of novel proteins Mass spectrometry (MS)-based global quantification of protein expression and PTMs in biological samples using stable isotopes [e.g. Isotope-coded affinity tags (ICAT), stable isotope labeling with amino acids in cell culture (SILAC), dimethyl labeling] Absolute quantification of proteins using stable isotopically labeled standards Structural elucidation of small molecules (e.g. metabolites) using liquid chromatography - accurate mass measurements MS analysis of chemically cross-linked peptides Method development for proteomics (e.g. chemical derivatization of PTMs) Instrumentation: Linear Ion Trap (LTQ) -Orbitrap Mass Spectrometer coupled to nanoscale liquid chromatography Two Linear Ion Trap (LTQ) Mass Spectrometers coupled to nanoscale liquid chromatography Personnel: Dr. Ying Wai Lam, Director (left) Tel: (802) 656-4709; E-mail: [email protected] Dr. Bin Deng, Manager (middle) Tel: (802) 656-5099; E-mail: [email protected] Julia Fields, Technician (right) Tel: (802) 656-1186; E-mail: [email protected] F ACILITY GUIDELINES Sample Submission, and Data Management T YPICAL W ORKFLOW OF B OTTOM - UP P ROTEOMICS Protein identification Step 1: Initial consultation: Investigators should contact the facility prior to sample submission to ensure a clear understanding of sample requirements for processing through the facility. To arrange a consultation, online consultation signups are available on our website or you can contact Drs. Wai Lam / Bin Deng by phone (802-656-4709, 802-656-5099) or email ([email protected], [email protected]). Step 2: Sample preparation: For gel samples, please provide an image of the gel and indicate the bands or spots that are to be analyzed. We need to be sure that your samples and sample preparation methods are compatible with mass spectrometry and that your sample has an adequate amount of the target protein(s) to be measured by mass spectrometry. Usually, a visible silver or Coomassie Blue-stained gel band/spot that corresponds to 20 femtomole - 1 picomole of protein is needed to identify the protein. PTM Analysis Quantitative Proteomics Using Stable Isotopes For protein samples in solution, you must provide the expected protein concentration and composition of the solution. Samples submitted for phosphorylation analysis must be Coomassie -stainable (in gel) or with a concentration higher than 5 pmole (in solution). Step 3: Fill out the sample submission form and answer the questions listed: Each sample must be accompanied by a completed form — please go to our website for online submission. The information you provide will help us to ensure proper handling and analysis of your samples. Step 4: Analysis: Only trained personnel directly operate the mass spectrometers in the facility. However, users are welcome to learn and participate in data analysis. Step 5: Results: Results will be sent via e-mail or provided as a printed copy. Raw data will be provided upon request. It is best to send your samples via overnight shipping if you are a non-UVM user, otherwise please drop them off in person. Please note that drop-off hours are Monday through Friday, 9:00 AM to 4:00 PM, and you are encouraged to give us a call before dropping off. If you have any questions, please feel free to contact us! The higher the quality of your samples , the quicker you will receive your successful results!!! Metabolite Identification