Characterization Of Proteins By Mass Spectrometry

Child Health And Human Development

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Abstract

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?" Three mass spectrometric approaches are available for this effort. Matrix Assisted Laser Desorption Ionization (MALDI) with Time-of-Flight (TOF) mass analysis, liquid chromatography (LC) followed by electrospray ionization with mass analysis in an instrument capable of using fragmentation reactions to generate peptide sequences, i.e. LC-MS/MS , and most recently MALDI followed by tandem TOF analysis for the determination of peptide sequences from fragment ion spectra. The latter method state of the art technology has become available during this year and has been demonstrated to provide identifications of mixtures of proteins at levels of about 100 fmole . With this combination of instrumentation, we are confident that, given enough material in a gel band to allow as much as 100 fmole to be available for analysis, 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 order to improve protein characterization 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 incompleteness or error. We are taking the approach termed complete de novo sequencing of peptides. This approach requires detailed interpretation of individual fragmentation mass spectra of peptides and is being implemented in conjunction with software designed and written in this Section. The approach has been applied to date to the sequencing of peptides from a series of standard tryptic digests as well as for tryptic digests of proteins isolated from sea urchin cortical vesicles. Results to date have shown that not only are the correct sequences found by the program as the highest scoring possibility from a spectrum, but that the spectra allow Leu residues to be distinguished from Ile. In a closely related area, we have made substantial progress in the development of a consistent methodology for the identification of site specific phosphorylation. A combination of following neutral phosphate losses in collision induced fragmentation (CID)under LC-MS/MS conditions to identifiy single phosphorylations coupled with the ability to identify multiple phosphorylations on single peptides under conditions of non-CID conditions with the MALDI TOF/TOF appears to hold considerable promise.