Combining Microseparations and Ion Mobility Mass Spectrometry to Probe Peptidergic Signaling in Environmental Stress

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Professor Lingjun Li of the University of Wisconsin at Madison is supported by the Chemical Measurement and Imaging Program in the Division of Chemistry to develop multi-faceted analytical methodologies, largely based on ion mobility mass spectrometry (IM-MS) and microseparation techniques for large-scale functional discovery, quantitation and structural elucidation of neuropeptides in a crustacean model system. Peptides play a key role in various physiological processes and the selected crab model is an ideal platform on which to develop novel mass spectral techniques and apply these techniques to gain biologically relevant information. The project findings are expected to expand our knowledge base in neuropeptide family organization and functional consequences of neuropeptide multiplicity in these important model organisms, with a focus on response to environmental stress. In this project, studies are being conducted to develop multi-faceted ion mobility mass spectrometry (IM-MS) and microseparation techniques for the separation and analysis of neuropeptides. The project has four separate but related specific aims: (1) to develop a multi-dimensional separation platform based on pairing capillary electrophoresis (CE) with electrospray ionization ion mobility-mass spectrometry (ESI-IM-MS), and use it for the analysis of neuropeptides; (2) to develop an ion mobility- mass spectrometry (IM-MS)-assisted isobaric tagging quantitation method and apply it to the study of peptidomic changes in response to environmental stress; (3) to develop an IM-MS-based site-specific strategy for rapid and accurate localization of D-amino acids in peptide epimers; and (4) to develop a strategy based on IM-MS and electron-transfer dissociation (ETD) to study the mechanism of gas phase peptide sequence scrambling and its impact on peptide de novo sequencing and to construct a collision cross section database to facilitate endogenous neuropeptide identification. During the course of conducting this project, a diverse group of students will be trained in advanced mass spectrometry, analytical separation methodologies and neuroscience. A partnership with the Madison Metropolitan School District program to host and mentor high school students for summer research internships will be continued and a web-based peptidome database system will be constructed.