Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India.
Anal Chem. 2010 Oct 1;82(19):8313-9. doi: 10.1021/ac101867e.
The critical, and often most difficult, step in structure elucidation of diverse classes of natural peptides is the determination of correct disulfide pairing between multiple cysteine residues. Here, we present a direct mass spectrometric analytical methodology for the determination of disulfide pairing. Protonated peptides, having multiple disulfide bonds, fragmented under collision induced dissociation (CID) conditions and preferentially cleave along the peptide backbone, with occasional disulfide fragmentation either by C(β)-S bond cleavage through H(α) abstraction to yield dehydroalanine and cysteinepersulfide, or by S-S bond cleavage through H(β) abstraction to yield the thioaldehyde and cysteine. Further fragmentation of the initial set of product ions (MS(n)) yields third and fourth generation fragment ions, permitting a distinction between the various possible disulfide bonded structures. This approach is illustrated by establishing cysteine pairing patterns in five conotoxins containing two disulfide bonds. The methodology is extended to the Conus araneosus peptides Ar1446 and Ar1430, two 14 residue sequences containing 3 disulfide bonds. A distinction between 15 possible disulfide pairing schemes becomes possible using direct mass spectral fragmentation of the native peptides together with fragmentation of enzymatically nicked peptides.
在不同类型天然肽的结构阐明中，最关键且通常也是最困难的步骤是确定多个半胱氨酸残基之间正确的二硫键配对。在这里，我们提出了一种直接的质谱分析方法来确定二硫键配对。带多个二硫键的质子化肽在碰撞诱导解离（CID）条件下发生碎片化，并且优先沿着肽骨架断裂，偶尔也会发生二硫键断裂，通过 H(α) 夺取生成脱氢丙氨酸和半胱氨酸过硫化物，或者通过 H(β) 夺取生成硫代醛和半胱氨酸，从而发生 C(β)-S 键断裂。初始产物离子（MS(n)）的进一步碎片化产生第三和第四代碎片离子，从而可以区分各种可能的二硫键结合结构。这一方法通过确定含有两个二硫键的五个 Conotoxin 中的半胱氨酸配对模式得到了验证。该方法还扩展到了 Conus araneosus 肽 Ar1446 和 Ar1430，这两种 14 个残基的序列含有 3 个二硫键。通过直接对天然肽进行质谱碎片化以及对酶切肽进行碎片化，可以区分 15 种可能的二硫键配对方案。
