The principal goal of this facility is microscale identification and detailed characterization of proteins and peptides using mass spectrometry. In the past fiscal year,numerous 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?"""""""" Towards the end of this period, the capabilities of the operation were expanded appreciably by the addition of an LC/MS instrument capable of using fragmentation reactions to determine peptide sequences, i.e. MS/MS and MS/MS/MS. In addition, this new instrumentation has been operated in a low LC flow rate mode that exhibits a >100-fold increase in sensitivity relative to commonly used micro-bore flow rates for typical peptide test mixtures. 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 few picomoles of protein. We expect that the new instrumentation will extend this capability. The success rate of identification gel separated proteins that have been reported in a data base remains >70%. Other results include the development of a novel analytical approach to the characterization of mixtures of proteins of very similar mass that cannot be otherwise distinguished. The approach requires no additional instrumentation. The method developed employs analysis of normalized cumulative distribution functions (NCUD) generated from the protonated parent ions, MH+, of the MALDI mass spectra of intact proteins. The maximum difference between the NCUDs of different, but closely related proteins is compared to a critical value of the Kolmogorov-Smirnov non-parametric statistic. Using standard acceptance criteria for rejection of the statistical null hypothesis, protein spectra that differ from one another, without appearing to do so, are identified. The method has been used successfully to resolve ambiguities for a number of otherwise indistinguishable proteins. Patent protection for this novel approach is being pursued by the Institute.
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