CNBr/formic acid reactions of methionine- 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-type and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed analysis of the mass spectra of reverse-phase HPLC-purified cleavage fragments produced from treatment of the wild-type and labeled proteins with CNBr indicated cleavage solely of methionyl peptide bonds with no observation of cleavage at TFM. N-Acetyl-TFM was also found to be resistant to reaction with CNBr, in contrast to N-acetyl-methionine. The analysis also indicated differential reactivity among the three methionine positions in the wild-type enzyme. Additionally, formylation of intact enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserine side chains are partially formylated under standard cleavage protocols.
Duewel
H S
HS
Department of Chemistry, University of Waterloo, Ontario, Canada.
Honek
J F
JF
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology; D003488:Cyanogen Bromide / Q000737:chemistry*; D005561:Formates / Q000737:chemistry*; D007202:Indicators and Reagents; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005561:Formates; D007202:Indicators and Reagents; D015335:Molecular Probes; D011994:Recombinant Proteins; C030544:formic acid; C037646:trifluoromethionine; D008715:Methionine; D009113:Muramidase; D003488:Cyanogen Bromide