Duewel H S, Honek J F
Department of Chemistry, University of Waterloo, Ontario, Canada.
J Protein Chem. 1998 May;17(4):337-50. doi: 10.1023/a:1022555232364.
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.
利用电喷雾电离（ESI）和基质辅助激光解吸电离（MALDI）质谱法研究了野生型和含三氟甲硫氨酸（TFM）的重组λ溶菌酶的溴化氰（CNBr）/甲酸裂解反应。对用CNBr处理野生型和标记蛋白后通过反相高效液相色谱（HPLC）纯化的裂解片段的质谱进行详细分析，结果表明仅甲硫氨酰肽键发生裂解，未观察到TFM处的裂解。与N-乙酰甲硫氨酸相反，还发现N-乙酰-TFM对与CNBr的反应具有抗性。分析还表明野生型酶中三个甲硫氨酸位置的反应性存在差异。此外，观察并表征了完整酶以及肽片段的甲酰化情况，结果表明在标准裂解方案下，丝氨酸、苏氨酸以及C末端高丝氨酸侧链会部分甲酰化。
