Novak Petr, Haskins William E, Ayson Marites J, Jacobsen Richard B, Schoeniger Joseph S, Leavell Michael D, Young Malin M, Kruppa Gary H
Sandia National Laboratories, Livermore, CA 94551, USA.
Anal Chem. 2005 Aug 15;77(16):5101-6. doi: 10.1021/ac040194r.
Fourier transform tandem mass spectrometry (FT-MS/MS) can be used to unambiguously assign intramolecular chemical cross-links to specific amino acid residues even when two or more possible cross-linking sites are adjacent in the cross-linked protein. Bovine rhodopsin (Rho) in its dark-adapted state was intramolecularly cross-linked with lysine-cysteine (K-C) or lysine-lysine (K-K) cross-linkers to obtain interatomic distance information. Large, multiply charged, cross-linked peptide ions containing adjacent lysines, corresponding to Rho(50-86) (K(66) or K(67)) cross-linked to Rho(310-317) (C(316)) or Rho(318-348) (K(325) or K(339)), were fragmented by collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD). Complementary sequence-specific information was obtained by combining cross-link assignments; however, only ECD revealed full palmitoylation of adjacent cysteines (C(322) and C(323)) and cross-linking of K(67) (and not K(66)) to C(316), K(325), and K(339). ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K(66) and K(67). To our knowledge, this work also presents the first demonstration that ECD can be used to characterize S-linked fatty acid acylation on cysteines. The comprehensive fragmentation of large peptides by CID, IRMPD, and particularly ECD, in conjunction with the high resolution and mass accuracy of FT-MS/MS, is shown to be a valuable means of characterizing mammalian membrane proteins with both chemical and posttranslational modifications.
傅里叶变换串联质谱法（FT-MS/MS）可用于明确将分子内化学交联指定到特定氨基酸残基，即使在交联蛋白中有两个或更多可能的交联位点相邻时也是如此。处于暗适应状态的牛视紫红质（Rho）与赖氨酸-半胱氨酸（K-C）或赖氨酸-赖氨酸（K-K）交联剂进行分子内交联，以获得原子间距离信息。含有相邻赖氨酸的大的、多电荷交联肽离子，对应于与Rho(310 - 317)（C(316)）或Rho(318 - 348)（K(325)或K(339)）交联的Rho(50 - 86)（K(66)或K(67)），通过碰撞诱导解离（CID）、红外多光子解离（IRMPD）和电子捕获解离（ECD）进行碎片化。通过组合交联归属获得了互补的序列特异性信息；然而，只有ECD揭示了相邻半胱氨酸（C(322)和C(323)）的完全棕榈酰化以及K(67)（而非K(66)）与C(316)、K(325)和K(339)的交联。ECD谱包含了由K(66)和K(67)之间的键断裂产生的关键c离子和z离子。据我们所知，这项工作还首次证明ECD可用于表征半胱氨酸上的S-连接脂肪酸酰化。CID、IRMPD，特别是ECD对大肽的全面碎片化，结合FT-MS/MS的高分辨率和质量准确性，被证明是表征具有化学修饰和翻译后修饰的哺乳动物膜蛋白的一种有价值的手段。
