CNBR FORMIC ACID REACTIONS OF METHIONINE-CONTAINING AND TRIFLUOROMETHIONINE-CONTAINING LAMBDA-LYSOZYME - PROBING CHEMICAL AND POSITIONAL REACTIVITY AND FORMYLATION SIDE REACTIONS BY MASS-SPECTROMETRY/

The cyanogen bromide (CNBr)/formic acid cleavage reactions of wild-typ e and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed anal ysis of the mass spectra of reverse-phase HPLC-purified cleavage fragm ents produced from treatment of the wild-type and labeled proteins wit h CNBr indicated cleavage solely of methionyl peptide bonds with no ob servation of cleavage at TFM. N-Acetyl-TFM was also found to be resist ant to reaction with CNBr, in contrast to N-acetyl-methionine. The ana lysis also indicated differential reactivity among the three methionin e positions in the wild-type enzyme. Additionally, formylation of inta ct enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserin e side chains are partially formylated under standard cleavage protoco ls.