Mass spectral characterization of organophosphate-labeled, tyrosine-containing peptides: Characteristic mass fragments and a new binding motif for organophosphates

We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8-8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the presentworkwere (1) to determine thecommonfeatures of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244amu for tyrosine-OP immonium ionswere nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared fromthe parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to findwhentheOPbears no radiolabel and no tag. The characteristicMSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.