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Analysis of organophosphorus compound adducts of serine proteases by liquid chromatography-tandem mass spectrometry.
Artigo em Inglês | MEDLINE | ID: mdl-12127328
ABSTRACT
In order to confirm that diisopropylfluorophosphate (DFP) phosphorylates the active site serine residue in alpha-chymotrypsin, a peptide containing the phosphorylated active site was analyzed by liquid chromatography (LC)-electrospray mass spectrometry (ESI-MS). After reduction with dithiothreitol and subsequent alkylation with acrylamide, alpha-chymotrypsin was digested by treatment with trypsin. Tryptic digest was subjected to LC-ESI-MS. Nearly all the peptide fragments were identified by comparison with fragments predicted from as tryptic digest of alpha-chymotrypsin. From the tryptic digest of native alpha-chymotrypsin, a doubly protonated peptide peak which corresponded to the peptide fragment containing the active site serine residue was detected on a selected ion chromatogram at m/z 1265.0, and the sequence was determined to be "DAMICAGASGVSSCMGDSGGPLVCK". From the tryptic digest of DFP-inhibited alpha-chymotrypsin, the doubly protonated peptide peak was detected on a selected ion chromatogram at m/z 1347.0. The difference in mass number (82 in a doubly charged ion) of active site peptide fragments between the native and DFP inhibited alpha-chymotrypsins was assumed to be the result of phosphorylation of the serine residue with a diisopropylphosphoryl moiety. A total of +164 Da mass shifts of y-series fragment ions from the y(8) to y(21) positions in the active site peptide of the DFP inhibited alpha-chymotrypsin was observed, in comparison with the native alpha-chymotrypsin. Thus, the phosphorylation site in alpha-chymotrypsin could be unequivocally identified to be at the serine residue which is located at position 47, from the N-terminus of the alpha-chymotrypsin C-chain.
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Coleções: Bases de dados internacionais Base de dados: MEDLINE Assunto principal: Compostos Organofosforados / Fragmentos de Peptídeos / Serina Endopeptidases / Cromatografia Líquida / Espectrometria de Massas por Ionização por Electrospray Aspecto clínico: Predição / Prognóstico Idioma: Inglês Revista: J Chromatogr B Analyt Technol Biomed Life Sci Assunto da revista: Engenharia Biomédica Ano de publicação: 2002 Tipo de documento: Artigo País de afiliação: Japão