Analysis of organophosphorus compound adducts of serine proteases by liquid chromatography–tandem mass spectrometry

Abstract

In order to confirm that diisopropylfluorophosphate (DFP) phosphorylates the active site serine residue in α-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, α-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 α-chymotrypsin. From the tryptic digest of native α-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 α-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 α-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 α-chymotrypsin was observed, in comparison with the native α-chymotrypsin. Thus, the phosphorylation site in α-chymotrypsin could be unequivocally identified to be at the serine residue which is located at position 47, from the N-terminus of the α-chymotrypsin C-chain.