The NBC Proteomics Core (PC) is located within 6 (i.e., Amino Acid Analysis, Biostatistics, Mass Spectrometry, Small and Large Scale Peptide Synthesis, and Protein Sequencing) of the 12 Resources that are the Keck Laboratory at Yale University. With 45 full time staff, including 12 with Ph.D. and 6 with M.S. level degrees, and >75 major instrument systems purchased at a cost of >$9 million dollars, the Keck Laboratory is one of the largest academic biotechnology resource laboratories of its kind. The PC provides a broad range of expertise and analytical capabilities to support basic science, preclinical, and clinical research programs in the NBC. In addition to providing proteomic services, the PC will also consult with members of the NBC regarding experimental design, optimum technology to best support the proposed research, and interpretation of proteomic data produced either by the PC or by other laboratories.
- “Traditional” proteomic services include ion exchange amino acid analysis, which is carried out on a Beckman 7300, and is most often used for accurately quantifying protein/peptide concentrations and for confirming the compositions of synthetic peptides. Amino-terminal (Edman) sequencing of peptides and proteins is carried out on two Applied Biosystems Procise 494 cLC protein sequencers which have a limit of detection of about 50 fmol of an individual phenylthiohydantoin amino acid. Providing that the N-terminus is not blocked, 1 to 10 pmol peptide/protein is sufficient to sequence from 5 to 25 or more residues. The quality of data and length of sequence that can be assigned increases with the amount of sample such that the best results are obtained on samples in the 10-100 pmol range. Samples should be submitted on PVDF membranes or in <0.1 ml of a volatile solvent such as water or 0.05% trifluoroacetic acid.
- The Mass Spectrometry Resource provides a comprehensive suite of protein mass spectrometry analyses which range from MALDI-MS based peptide/protein disease biomarker discovery to LC MS/MS protein identification to exact mass determinations on small molecules. Currently, LC-MS/MS spectra from tryptic digests of approximately 75% of "unknown" proteins meet the Keck criteria for a positive protein identification using the Mascot search algorithm. A new Applied Biosystems Q-Star mass spectrometer system is being used to provide isotope-coded affinity tag (ICAT)-based analysis of protein expression using the cleavable ICAT reagent from Applied Biosystems. ICAT/MS-based analysis of differential protein expression is nicely complemented by an Amersham differential (fluorescence) 2D gel electrophoresis (DIGE) system. DIGE technology, which is based on Cy-3/Cy-5 in vitro labeling of control/experimental protein extracts prior to mixing the two samples together and subjecting to 2D PAGE (in a single gel), has a dynamic range of 104 and is able to quantify the relative level of expression of 1,000 or more proteins/2D gel. Finally, a Bruker 9.4T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer, which will be installed in June, 2004 and will be equipped with both ESI and MALDI sources, is expected to be used for multi-dimensional protein identification technology (MudPit) and for identification of protein post-translational modifications.
- Small scale, Fmoc peptide synthesis is carried out on a Rainin Symphony instrument and is suitable for peptides that generally range from 7 to 30 or more residues in length. A wide range of modified amino acids may be incorporated into small scale synthetic peptides with the primary limitation being the commercial availability of the required Fmoc derivative. Peptides may be purchased either before or after RP-HPLC purification and are characterized by MALDI-MS and analytical reverse phase HPLC analysis. The Keck Large Scale Peptide Synthesis Resource utilizes tBOC synthesis with peptides being made routinely up to 40 residues and often, depending on sequence, up to 70 residues. All peptides that can be purified are chromatographed on a preparative C-18 or C-4 RP-HPLC system and delivered as a lyophilized material. Yields for normal peptides under 40 residues are "guaranteed" at 50 mg or more and at 90+% purity. For the incorporation of unusual amino acids, such as non-radioactive isotopes, the appropriate tBOC and side-chain protected material must be supplied by the submitter at a level of 2 mmol/residue. The purified peptides are characterized by analytical RP-HPLC, amino acid analysis, and MALDIMS.
- The Biostatistics Resource provides state-of-the-art statistical analyses to identify proteins with different expression levels among a set of conditions, to cluster proteins with co-regulated expression patterns, and to identify biomarkers for disease diagnosis and prognosis. In addition to assisting with the statistical analysis of isotope coded affinity tag (ICAT) and differential fluorescence (2D) gel electrophoresis (DIGE)- based protein expression data, the Biostatistics Resource has developed a customized Random Forest-based algorithm for disease biomarker discovery which currently relies on matrix assisted laser desorption ionization mass spectrometry analysis of serum (Wu et al (2003) Bioinformatics 19: 1636-1643). The Biostatistics Resource will assist with statistical analysis of proteomic data obtained either in the PC or in other laboratories and resources.
A listing of all Keck service charges is at: http://keck.med.yale.edu/price.htm. NBC members are given a 30% discount off the Yale or Non-Yale/Non-Profit charges for proteomic services providing they preface their Yale charging instructions or purchase order number with: NBC/. Submission forms and other information about requesting services from the Keck Laboratory may be found at: http://keck.med.yale.edu/ord_info.htm
Inquiries can be made to the director of each resource, with the directors listed along with their contact information at: http://keck.med.yale.edu/staff.htm; or by contacting:
Director of the Proteomics Core