Analysis of organophosphorus compound adducts of serine proteases by liquid chromatography-tandem mass spectrometry.
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.
Tsuge
Kouichiro
K
National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan. tuge@nrips.go.jp
Seto
Yasuo
Y
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000032:analysis*; D010446:Peptide Fragments / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D012697:Serine Endopeptidases