Goodlett D R, Armstrong F B, Creech R J, van Breemen R B
Department of Biochemistry, North Carolina State University, Raleigh 27695.
Anal Biochem. 1990 Apr;186(1):116-20. doi: 10.1016/0003-2697(90)90583-u.
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.
在溴化氰消化过程中，蛋白质暴露于70%的甲酸中，会在随后通过高效液相色谱（HPLC）纯化过程中导致假象峰的形成，并在快速原子轰击（FAB）质谱分析期间检测到[M + H + 28]+离子。溴化氰消化后，通过正离子FAB质谱分析了来自马心脏细胞色素c、细菌视紫红质、牛肾上腺髓质十二肽和牛肾上腺肽E的肽段。细胞色素c和细菌视紫红质的溴化氰肽段显示出甲酰化离子[M + H + 28]+和未甲酰化离子[M + H]+的混合物。牛肾上腺髓质十二肽和牛肾上腺肽E在消化过程中未被甲酰化。使用反相HPLC可将甲酰化肽段与相应的未甲酰化肽段分离。甲酰化肽段的不稳定性使得无法通过埃德曼降解确定加合物的位置。然而，在对细胞色素c的溴化氰肽段的[M + H + 28]+离子进行碰撞激活的FAB质谱分析过程中的B/E联动扫描表明，甲酰化发生在一个苏氨酸残基上。基于在水溶液中的稳定性测量和FAB质谱分析，确定在蛋白质溴化氰消化过程中，丝氨酸和苏氨酸残基是最有可能被甲酸酯化的位点。此外，在溴化氰消化过程中用70%的三氟乙酸替代甲酸可消除甲酰化，并且几乎不产生或不产生三氟乙酰化。
