The principal goal of this facility is the identification and detailed characterization of proteins and peptides at a microscale level using mass spectrometry. 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?"""""""" Both Matrix Assisted Laser Desorption Ionization (MALDI) with Time-of-Flight (TOF) mass analysis and LC followed by electrospray ionization with mass analysis in an instrument capable of using fragmentation reactions to generate peptide sequences, i.e. MS/MS , are used. The latter method has been implemented during this year and has been demonstrated to provide identifications of mixtures of proteins at levels of about 100 fmole injected onto packed capillary reverse phase columns operated at 400 nL/min flow rates. With the addition of this instrument to our facility, we are confident that, given enough material in a gel band to allow as much as 100 fmole to be applied to the LC column, a positive identification can be made for a protein that is described in a data base. There are two principal areas of development that are being carried out in oder to improve protein charachterization capabilities. First, we have begun addressing the question of providing sequence information on proteins that are not described in data bases, due either to data base incompletness or errror. We are taking the approach termed de novo sequencing of peptides. This approach requires detailed interpetation of individual mass spectra of peptides that have been subjected to fragmentation and mass analysis of the fragments. It is being implemented in our hands using a recently developed tandem TOF mass spectrometer in conjunction with interpretation software written in this Section. The approach has been applied to date to the sequencing of proteins isolated from sea urchin cortical vessicles and has been shown to be reasonably successful. Specifically, components of several bands isolated from the CV of sea urchins have been shown to have sequences that correspond to no known proteins, yet have homologies to other proteins that may provide insights into fusion processes. The second area of development involves the use of surface plasmon resonance detectors, as implemented by Biacore instruments, to isolate specific proteins. We have developed a model system in which we have demonstrated the isolation of 30 fmole of hen egg lysozyme in the presence of 30 pmoles of myoglobin. The isolated proteins are then recovered in small volumes so that concentrations are appropriate for mass spectrometric analysis. MALDI analysis of the recovered proteins shows strong signals from the isolated material and zero response from other proteins present.
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