Formylated peptides from cyanogen bromide digests identified by fast atom bombardment mass spectrometry.
Exposure of proteins to 70% formic acid during cyanogen bromide digestion can result in formation of artifact peaks during subsequent purification by HPLC and detection of [M + H + 28]+ ions during analysis by fast atom bombardment (FAB) mass spectrometry. Following cyanogen bromide digestion, peptides from equine heart cytochrome c, bacteriorhodopsin, bovine adrenal medulla dodecapeptide, and bovine adrenal peptide E were analyzed by positive ion FAB mass spectrometry. The cyanogen bromide peptides of cytochrome c and bacteriorhodopsin showed mixtures of formylated ions, [M + H + 28]+, and nonformylated ions, [M + H]+. Bovine adrenal medulla dodecapeptide and bovine adrenal peptide E were not formylated during digestion. Formylated peptides could be resolved from the corresponding nonformylated peptides using reversed-phase HPLC. Instability of the formylated peptides prevented localization of the adduct by Edman degradation. However, B/E-linked scanning during FAB mass spectrometric analysis with collisional activation of the [M + H + 28]+ ion of a cyanogen bromide peptide from cytochrome c suggested that formylation occurred at a threonine residue. On the basis of stability measurements in aqueous solution and analysis by FAB mass spectrometry, it was determined that serine and threonine residues are the most likely sites of esterification by formic acid during cyanogen bromide digestion of proteins. Furthermore, substitution of 70% trifluoroacetic acid for formic acid during cyanogen bromide digestion eliminated formylation and generated little or no trifluoroacetylation.
Goodlett
D R
DR
Department of Biochemistry, North Carolina State University, Raleigh 27695.
Armstrong
F B
FB
Creech
R J
RJ
van Breemen
R B
RB
D000313:Adrenal Medulla; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000378:metabolism*; D005561:Formates / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides; D014269:Trifluoroacetic Acid
D005561:Formates; D010455:Peptides; D014269:Trifluoroacetic Acid; D003488:Cyanogen Bromide