The principal goal of this facility is microscale identification and detailed characterization of proteins and peptides using mass spectrometry. In the past fiscal year, the samples for analysis have been generated through a series of collaborative interactions. For identification of unknown proteins, the MS data are used to query genomic databases to ask the general question, Do any of the protein sequences present in the data base have expected proteolytic cleavage products with theoretical masses that match the empirically determined masses of the peptides generated from the unknown? In addition, to addressing this and closely related set of problems, the facility undertakes development of methodologies to extend its capabilities. The facility has firmly established its capability for peptide fingerprinting of in situ digestion of PAGE separated proteins using the MALDI-TOF instrumentation at the level of a picomole of protein. Partial peptide sequences have been obtained using either HPLC- electrospray MS/MS or MALDI-TOF based PSD approach for a number of peptides. In general, in cases where samples provided are of molecules that have been reported in a data base as either amino acid or cDNA sequences, the success rate of identification is >70%. Other results include 1) verification of synthesis of carbohydrate units coupled to core globular protein (with R. Schneerson, LDMI). This work has been particularly successful and represents an advance in the ability to produce a synthetic vaccine for cholera. 2) Continued development of the use of Surface Plasmon Resonance (SPR) in conjunction with MALDI-TOF MS has been used in a) the elucidation of the structure of the extracellular domain of the human parathyroid calcium receptor (with P.Goldsmith, NIDDK) and with b) in the elucidation of the cell surface binding epitopes of the heavy chain region of botulism toxin as part of a program in developing a botulism vaccine (with T. Smith, USAMRID). 3) Characterization of tubulin molecules separated by isoelectric focussing followed by solvent based elution of excised gel bands (with D. Sackett, LIMB). - Proteins, Peptides, Identification, Mass Spectrometry
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