MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Department of Analytical Chemistry, ELTE Eötvös Loránd University, Budapest 1117, Hungary.
Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest 1117, Hungary.
J Am Soc Mass Spectrom. 2023 Aug 2;34(8):1569-1575. doi: 10.1021/jasms.3c00044. Epub 2023 Jul 6.
Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH/z, c, c-NH/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages -terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage -terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.
通过正电喷雾电离（ESI+）串联质谱（MS/MS）鉴定肽是蛋白质组学中一种成熟的策略。一些研究小组报告说，与 ESI+相比，负电喷雾电离（ESI-）在获得肽及其翻译后修饰（PTM）的补充结构信息方面非常有用。以前尚未在 ESI-中探索瓜氨酸化肽的碎裂。在这项研究中，通过 QTOF 仪器和 Q-Orbitrap 仪器的逐步碰撞能依赖性测量，研究了 9 个含有瓜氨酸残基的肽。我们的高分辨率和质量精度结果表明，这些肽前体及其片段中的瓜氨酸选择性 HNCO 丢失得到了有利的支持─与 ESI+中的情况类似─同时还有 y-NH/z、c、c-NH/b 序列离子。首次在这里描述了 ESI-中瓜氨酸化肽的 HNCO 丢失以及该反应的提议机制。与 ESI+相比，前体的 HNCO 丢失强度通常更高。有趣的是，与序列离子相关的最强碎片对应于中性损失，而完整的序列离子通常是谱图的次要成分。还观察到了以前报道过的与 Asp 和 Glu 残基 -末端的切割相关的高强度离子。另一方面，可能由于内部碎裂和/或重排事件，观察到相对较多的峰。虽然（ESI-）MS/MS 谱总是需要手动检查，并且注释可能存在歧义，但 HNCO 的有利丢失和 Asp 残基 -末端的优选切割可用于区分瓜氨酸化/去酰胺化序列。
