Research to solve a number of structural problems in problems in protein chemistry is proposed. Efforts will be made; (a) to elucidate the amino acid sequence of a novel iron containing purple acid phosphatase from beef spleen, (b) to determine the transmembrane arrangement of the acetylcholine receptor protein, (c) to identify and characterize the colchicine binding site on the protein, tubulin, in microtubules, (d) to sequence the Alpha-amlase used as a marker for serous ovarian tumors, and (e) to determine the amino acid sequence of iodopsin, the color vision cone protein from chicken. Newly developed methodology based on tandem mass spectrometry will be employed in these studies. This involves chemical and/or enzymatic degradation of the protein, fractionation of the resulting oligopeptides by HPLC, mol wt characterization of the individual components of each fraction by liquid secondary ion mass spectrometry, and sequence analysis of each mixture component by the technique of collision activated dissociation on a triple quadrupole mass spectrometer. The strengths of this approach are that it does not require highly purified sample, that it works well on proteins with blocked N-termini, and that it provides extensive sequence information over the whole protein chain at the 10 nmol level in a single 4 to 5 day experiment that involves minimal effort directed toward separation and purification of the oligopeptide mixtures. Research to continue the development of the tandem mass spectrometry methodology for sequence analysis of proteins is also proposed. Efforts will be made to extend the mass range of our triple quadrupole mass spectrometer beyond 3,00 amu, to lower the quantity of sample required to the subnanomole level, to develop an approach for selective identification and sequence analysis of C-terminal protein fragments that can be used to direct the synthesis of oligonucleotide probes, to develop an approach for the selective identification and sequence analysis of N-terminal protein fragments, and to explore derivatization procedures that minimize sample ion suppression and maximize the formation of fragment ions carrying sequence information during collision activated dissociation experiments.
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