PROTEIN SEQUENCING BY TANDEM MASS SPECTROMETRY

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Research to solve a number of structural problems in protein chemistry and to continue the development of instrumentation and methodology for the sequence analysis of proteins by tandem mass spectrometry is proposed. Efforts will be made; (a) to develop spectrometry for the direct sequence analysis of disease diagnostic proteins isolated at the femtomole/picomole level from cultured cells and biological fluids by two dimensional gel electrophoresis, (b) to characterize the structural modifications that proteins in the human lens undergo during cataract formation, (c) to use the laser photocrosslinking technique in combination with tandem mass spectrometry to pinpoint amino acid residues in DNA polymerase I involved in both the binding domain at which synthesis proceeds and the deoxynucleotide triphosphate binding site where the next complementary nucleotide is held, (d) to establish the primary structures of two calcium binding proteins involved in neuronal differentiation in the brain, (e) to sequence the regulatory light chain isoforms of myosin involved in maintaining high blood pressure in vascular smooth muscle, (f) to characterize the topography or folding pattern of the nicotinic acetylcholine receptor protein that makes up the ligand gated ion channel at the neuromuscular junction, (g) to locate the nine phosphorylated residues in the visual protein, rhodopsin, and to determine the order in which these residues are phosphorylated as a first step in understanding the role that phosphorylation plays in regulation of the visual process at the molecular level, (h) to establish the primary structures of human monocyte chemoattractant proteins involved in modulating the immune response to antigens, and (i) to sequence aminoacylase and two phosphotyrosyl phosphatases involved in cell proliferation. Efforts will also be made to reduce the amount of sample required for analysis on the newly acquired Model 270 triple quadrupole mass spectrometer to the low picomole range and to extend the usable mass range of this instrument beyond m/z 3,000. Capillary electrophoresis will be interfaced to the triple quadrupole and quadrupole FTMS instruments via electrospray ion sources to facilitate online analysis of protein and oligopeptide samples separated by this extremely powerful technique. Fragmentation of multiply charged peptide ions will be studied and conditions that allow differentiation of leucine and isoleucine residues by the process of collision activated dissociation or surface induced dissociation will be explored.