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.
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