TANDEM ON/OFF EXCITATION OF IONS IN VIBRATIONALLY COOLED MALDI FTICR MS

振动冷却 MALDI FTICR MS 中离子的串联开/关激发

• 批准号: 7369271
• 负责人: PETER B. O'CONNOR
• 金额: $ 1.43万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369271
• 关键词: TANDEM; OFF; EXCITATION; IONS; VIBRATIONALLY

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Vibrational Cooling (VC) MALDI-FTMS generates ions by desorbing/ionizing analytes under 1-10 mbar of collision gas. This method allows for detection of labile species with FTMS resolution and accuracy, making this instrument highly applicable for proteomic experiments, particularly for the analysis of labile post-translational modifications (PTM?s). However, once the ions are generated, it is necessary to perform MS/MS experiments on them. This presentation concerns investigation of use of multiple collisional activation events as a tandem mass spectrometry method which provides nearly full sequence coverage for these peptides. VC MALDI FTMS has previously been described [1]. In the described experiments, multishot accumulation was used to acquire high abundance of ions in the hexapole. Subsequently ions were transferred to the cell, where precursor ions were isolated by SWIFT. These ions were then subjected to a series of on- and off- resonant excitation events similar to MECA technique [2] and SORI [ref Jacobsen]. Synthetic peptides were used to probe the fragmentation of unmodified and post-translationally modified peptides with varying lability. 1. O'Connor PB, Mirgorodskaya E, Costello CE JASMS 2002, 13, 402-407 2. Lee SA, Jiao CQ, Huang YQ, Freiser BS RCM 1993, 7(9), 819-821 Experiments using a series of SORI- and MECA- type collisional activation events were performed on peptides formed by VC MALDI. As expected, SORI-CAD of these peptides generated a few specific cleavages. However, use of multiple SORI and MECA events for activation yielded high quality CAD spectra of these peptides, generating b/y type cleavage at almost every peptide bond. Furthermore, these CAD spectra when generated on the VC-MALDI-FTMS instrument preserved the information regarding the position of labile post-translational modifications such as phosphorylation and o-glycosylation. The same peptides, when analyzed with ESI-FTMS using the same multi-CAD approach, tended to preferentially lose the labile PTM and lose the positional information. A number of synthetic peptides were analyzed were analyzed to confirm this unexpected observation. For example, VC-MALDI-FTMS ACTH 18-39 ions under multi-CAD conditions, produced 10 b- and 10 y- type of fragments providing sequence information for 18 out of 21 amino acid residues; whereas the ESI-FTMS multi-CAD experiment, produced only 8 fragment ions in total. More interestingly, when applying multi-CAD to peptides with labile side chain modifications such as O-glycosylation on Ser and/or Thr residues, and single and multiple phosphorylation on Ser and Thr, information-rich spectra are generated. Most of the fragment ions in the product ion spectra still carry the labile side chain modification(-s) providing nearly complete sequence information and unambiguous localization of the sites of modifications. Ongoing work is investigating the application of this technique to sulfated peptides and carbohydrates which are normally extremely difficult to analyze by CAD-based MS/MS methods.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。振动冷却(VC)MALDI-FTMS通过在1-10毫巴的碰撞气体下解吸/电离分析物来产生离子。这种方法能够以FTMS的分辨率和准确性检测不稳定的物种，使该仪器非常适用于蛋白质组学实验，特别是分析不稳定的翻译后修饰(Ptm？S)。然而，一旦离子产生，就有必要对它们进行MS/MS实验。本报告涉及使用多个碰撞激活事件作为串联质谱学方法的研究，该方法提供了这些多肽的几乎全序列覆盖。VC MALDI FTMS以前已经描述过[1]。在所描述的实验中，使用多次激发积累来获得六极中的高丰度离子。随后，离子被转移到细胞，在那里，前体离子被SWIFT分离。然后，这些离子受到一系列与MECA技术[2]和Sori[参考Jacobsen]类似的开启和关闭共振激发事件的影响。合成的多肽被用来探索具有不同稳定性的未修饰和翻译后修饰的多肽的碎裂。1.O‘Connor PB，Mirgorodskaya E，Costello CE JASMS 2002，13,402-407 2.Lee SA，Jiao CQ，Huang YQ，Freiser BS RCM 1993，7(9)，819-821使用一系列Sori和MECA类型的碰撞活化事件对VC MALDI形成的多肽进行了实验。正如预期的那样，这些多肽的Sori-CAD产生了一些特定的裂解。然而，使用多个Sori和MECA事件进行激活可以获得这些多肽的高质量CAD谱，几乎在每个多肽键上都产生b/y型裂解。此外，当在VC-MALDI-FTMS仪器上生成这些CAD谱时，保存了关于不稳定的翻译后修饰的位置的信息，例如磷酸化和o-糖基化。相同的多肽，当使用相同的多CAD方法进行ESI-FTMS分析时，倾向于优先丢失不稳定的PTM并丢失位置信息。分析了许多合成肽，以证实这一意想不到的观察结果。例如，VC-MALDI-FTMS ACTH 18-39离子在多CAD条件下产生了10个b型和10个y型片段，提供了21个氨基酸残基中的18个的序列信息；而ESI-FTMS多CAD实验总共只产生了8个片段离子。更有趣的是，当对具有不稳定侧链修饰的多肽进行多重CAD时，例如丝氨酸和/或苏氨酸残基上的O-糖基化，以及丝氨酸和苏氨酸上的单一和多重磷酸化，就会产生信息丰富的光谱。产物离子谱中的大部分碎片离子仍然带有不稳定的侧链修饰(-S)，提供了几乎完整的序列信息和修饰位点的明确定位。正在进行的工作是研究将这项技术应用于硫酸基多肽和碳水化合物，这些物质通常很难用基于CAD的MS/MS方法进行分析。