Smith Lori L, Herrmann Kristin A, Wysocki Vicki H
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
J Am Soc Mass Spectrom. 2006 Jan;17(1):20-8. doi: 10.1016/j.jasms.2005.06.016. Epub 2005 Dec 9.
Unusual fragmentation was observed for doubly charged VPDPR in which cleavage C-terminal to proline and N-terminal to aspartic acid yielded b(2) (+ a(2))/y(3) complementary ions. This unique fragmentation is contradictory to trends previously established by statistical analysis of peptide tandem mass (MS/MS) spectra. Substitution of alanine for aspartic acid (i.e., VPAPR) did not change the fragmentation, indicating the cleavage was not directed by aspartic acid. Fragmentation patterns for VPAPR and V(NmA)APR (NmA = N-methyl alanine) were compared to determine whether conformational constraints from proline's cyclic side-chain contribute to b(2) ion formation. While both peptide sequences fragmented to yield b(2)/y(3) ions, only VPAPR produced a(2) ions, suggesting the VP b(2) ion is structurally different from the V(NmA) b(2) ion. Instead, the V(NmA) b(2) ion was accompanied by an ion corresponding to formal loss of 71. The suspected structural differences were confirmed by isolation and fragmentation of the respective b(2) ions (i.e., MS(3) spectra). Evidence supporting a diketopiperazine structure for the VP b(2) ion is reported. Fragmentation patterns for the VP b(2) ion and a synthetic VP diketopiperazine showed great similarity. N-terminal acetylation of VPAPR prevented the formation of the VP b(2) ion, presumably by blocking nucleophilic attack by the N-terminal amine on the carbonyl oxygen of the protonation site. Acetylation of the N-terminus for V(NmA)APR did not prevent the formation of the V(NmA) b(2) ion, indicating the V(NmA) b(2) ion has a structure, presumably that of an oxazolone, which requires no attack by the N-terminus for formation. Finally, high-resolution, accurate mass measurements determined that the V(NmA) (b(2)-71) ion results from losing a portion of valine from oxazolone V(NmA) b(2) ion, rather than cross-ring cleavage of the alternate diketopiperazine.
观察到双电荷VPDPR出现异常裂解，其中脯氨酸C端和天冬氨酸N端的裂解产生了b(2) (+ a(2))/y(3)互补离子。这种独特的裂解与先前通过肽串联质谱（MS/MS）谱统计分析确立的趋势相矛盾。用丙氨酸取代天冬氨酸（即VPAPR）并没有改变裂解情况，这表明裂解并非由天冬氨酸引导。比较了VPAPR和V(NmA)APR（NmA = N-甲基丙氨酸）的裂解模式，以确定脯氨酸环状侧链的构象限制是否有助于b(2)离子的形成。虽然两个肽序列裂解后都产生了b(2)/y(3)离子，但只有VPAPR产生了a(2)离子，这表明VP b(2)离子在结构上与V(NmA) b(2)离子不同。相反，V(NmA) b(2)离子伴随着一个对应于71正式丢失的离子。通过分离和裂解各自的b(2)离子（即MS(3)谱）证实了疑似的结构差异。报告了支持VP b(2)离子二酮哌嗪结构的证据。VP b(2)离子和合成的VP二酮哌嗪的裂解模式显示出极大的相似性。VPAPR的N端乙酰化阻止了VP b(2)离子的形成，大概是通过阻断N端胺对质子化位点羰基氧的亲核攻击。V(NmA)APR的N端乙酰化并没有阻止V(NmA) b(2)离子的形成，这表明V(NmA) b(2)离子具有一种结构，大概是恶唑酮的结构，其形成不需要N端的攻击。最后，高分辨率、精确质量测量确定V(NmA) (b(2)-71)离子是由于恶唑酮V(NmA) b(2)离子丢失了一部分缬氨酸，而不是交替二酮哌嗪的跨环裂解。
