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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。振动冷却（VC）MALDI-FTMS通过在1-10 mbar的碰撞气体下解吸/电离分析物来产生离子。这种方法允许检测不稳定的物种与FTMS分辨率和准确性，使该仪器高度适用于蛋白质组学实验，特别是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法，提供了几乎完整的序列覆盖这些肽的调查。 VC MALDI FTMS先前已描述[1]。在所描述的实验中，使用多次累积以在六极中获得高丰度的离子。随后，将离子转移到池中，在池中通过SWIFT分离前体离子。然后，这些离子经受一系列与MECA技术[2]和SORI [参考Jacobsen]类似的开和关共振激发事件。使用合成肽来探测具有不同不稳定性的未修饰和后修饰肽的片段化。 1.奥康纳PB，Mirgorodskaya E，Costello CE JASMS 2002，13，402-407 2. LeeSA，JiaoCQ，HuangYQ，FreiserBSRCM 1993，7（9），819-821使用一系列SORI和MECA型碰撞活化事件对由VCMALDI形成的肽进行实验.正如预期的，这些肽的SORI-CAD产生了一些特异性裂解。然而，使用多个SORI和MECA事件进行活化产生了这些肽的高质量CAD谱，在几乎每个肽键处产生B/y型裂解。此外，当在VC-MALDI-FTMS仪器上生成时，这些CAD光谱保留了关于不稳定的翻译后修饰（例如磷酸化和o-糖基化）的位置的信息。当使用相同的多CAD方法用ESI-FTMS分析时，相同的肽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，以证实这一意外观察结果。例如，VC-MALDI-FTMS ACTH 18-39离子在多CAD条件下产生10个B-和10个y-型片段，提供21个氨基酸残基中的18个的序列信息;而ESI-FTMS多CAD实验总共仅产生8个片段离子。 更有趣的是，当将多CAD应用于具有不稳定侧链修饰的肽时，例如Ser和/或Thr残基上的O-糖基化，以及Ser和Thr上的单磷酸化和多磷酸化，产生了信息丰富的光谱。产物离子光谱中的大多数碎片离子仍然携带不稳定的侧链修饰（-s），提供了几乎完整的序列信息和修饰位点的明确定位。 正在进行的工作是调查这项技术的应用，硫酸化肽和碳水化合物，通常是非常困难的CAD为基础的MS/MS方法进行分析。

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.