Laboratory of Mass Spectrometry, GIGA-R, Department of Chemistry, University of Liege, Liege, Belgium.
Anal Chem. 2013 May 7;85(9):4405-13. doi: 10.1021/ac303686w. Epub 2013 Apr 10.
Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using tandem mass spectrometry (MS/MS) spectra of the totally reduced unfolded species, but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of nonclassical ions. MS/MS alone does not allow either the cysteine pairing or the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining ion mobility spectrometry (IMS) and collision-induced dissociation (CID). It is assumed that the opening of one S-S bridge in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation, or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteine connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference.
二硫键在确定肽和蛋白质的结构特性方面起着重要作用。然而，确定半胱氨酸配对仍然具有挑战性。肽序列通常是使用完全还原的未折叠物种的串联质谱 (MS/MS) 谱来确定的，但半胱氨酸配对信息会丢失。另一方面，对天然折叠物种进行的 MS/MS 实验显示出由非经典离子组成的复杂光谱。MS/MS 本身既不能确定半胱氨酸配对，也不能确定未知肽的完整序列。这项工作的主要目标是建立一种策略，用于全面表征肽，包括在序列中标注二硫键。该策略是通过结合离子淌度谱 (IMS) 和碰撞诱导解离 (CID) 来开发的。假设肽中二硫键的打开会导致结构演变，从而导致 IMS 漂移时间发生变化。在存在多个二硫键的情况下，到达时间的偏移将取决于已还原的哪个（哪些）二硫键以及生成的物种所采用的形状。由于每种物种都观察到特定的碎片化，在淌度分离后进行的 CID 实验不仅可以提供肽序列的信息，还可以提供二硫键定位的信息。为了实现这一目标，研究了含有两个二硫键的合成肽。根据不同的实验条件（如还原、还原/烷基化或氧化）进行桥的打开。由于还原方法中突出的二硫键重排，将 S-S 键氧化成半胱氨酸酸似乎是最佳策略。然后，无需任何二硫键重排干扰，就可以明确确定这两个肽的半胱氨酸连接性。
