Tsaprailis George, Nair Hari, Zhong Wenqing, Kuppannan Krishnamoorthy, Futrell Jean H, Wysocki Vicki H
Department of Chemistry, University of Arizona, P.O. Box 210041, Tucson, AZ 85721-0041, USA.
Anal Chem. 2004 Apr 1;76(7):2083-94. doi: 10.1021/ac034971j.
Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.
研究了组氨酸附近肽段的气相裂解增强情况。所检测的肽段包括血管紧张素III（RVYIHPF）和IV（VYIHPF）以及关键残基改变的合成肽类似物（(R)VYI-X-Z-F；X = F或H且Z = A、P或Sar）或带固定电荷的M3P(+)CH(2)C(O)-VYIHPF。虽然所有同时含有组氨酸和精氨酸的单质子化肽离子无选择性地裂解，但含有精氨酸和组氨酸的双质子化肽离子以及含有组氨酸但不含精氨酸的单质子化肽以选择性方式裂解。特别是，观察到了由HP酰胺键之间裂解产生的主要互补b+/y+产物离子。对于带固定电荷的衍生物，只有在添加一个质子以产生双电荷前体时才会发生选择性裂解。结果与质子化组氨酸参与选择性裂解一致。b+/y+的比例由组氨酸C端残基的身份以及质子化组氨酸将质子转移至C端离去片段的能力决定。通过系统改变组氨酸C端的残基并对组氨酸进行烷基化进一步探究了这一点。结果表明，虽然双质子化的RVYIHPF中b+/y+互补离子对占主导，但双质子化的RVYIHAF的质谱中b5(2+)和b6(2+)产物离子占主导。此外，当双质子化的RVYIHPF中组氨酸侧链被烷基化（H）时，也观察到了占主导的b5(2+)产物离子。基于所有结果，提出了一种涉及非典型b离子结构的组氨酸增强裂解的选择性碎片化机制。
