"Development of a Multi-faceted Mass Spectrometry-based Platform for Neuroscience and Biomedical Research"
Professor Lingjun Li, University of Wisconsin-Madison
Comprehensive characterization of all signaling molecules in a nervous system with chemical, spatial and temporal information is often critical to deciphering the functionality of a neural circuit yet it presents a daunting challenge. In this presentation, I will present our recent progress on the development of a multi-faceted mass spectrometry (MS)-based analytical platform to probe neuronal signaling with enhanced sensitivity and selectivity. By combining chemical labeling, micro-scale separation, and tandem MS sequencing techniques, we discovered more than 300 novel neuropeptides in crustacean nervous systems and explored peptidomic changes in feeding using both crustacean and mammalian model organisms. Moreover, both mass spectrometric imaging (MSI) technology and in vivo microdialysis sampling tools have been developed and implemented to follow neuropeptide distribution and secretion with unprecedented details. Furthermore, novel dimethylated leucine (DiLeu) isobaric tagging reagents have been developed and employed to offer cost-effective implementations that enable higher order of multiplexing. Additionally, we report on a multiplexed quantitation method for simultaneous proteomics and amine metabolomics analyses via nanoflow reversed phase LC-MS/MS, exploiting mass defect-based DiLeu (mdDiLeu) labeling. Several on-going efforts and future perspectives provided by these enabling technologies will be highlighted and discussed.
"Sequential Enrichment of Phosphorylation on Reversibly Oxidized Proteoforms"
Evan McConnell, Laboratory of Professor Leslie Hicks, University of North Carolina-Chapel Hill, Department of Chemistry
Post-translational modifications (PTMs) are covalent modifications to the primary structure of proteins and are necessary for the regulation of critical processes including metabolism, signaling, and overall homeostasis. While PTMs have been largely investigated independently, examination into how different PTMs interact, or crosstalk, will reveal a more complete understanding of the reciprocity of signaling cascades across numerous pathways. Combinatorial reversible thiol oxidation and phosphorylation in eukaryotes is largely recognized, but rigorous approaches for experimental verification are underdeveloped and must be advanced to begin meaningful definition of crosstalk in targeted pathway and systems biology research. Herein, we applied protein-level enrichment of reversibly oxidized proteoforms from the photoautotrophic algae Chlamydomonas reinhardtii with subsequent phosphopeptide analysis to determine the extent of phosphorylation in the redox thiol proteome. Reversibly oxidized proteins are first enriched intact from the proteome, where additional enrichment of PTMs from previously bound proteins necessitates some degree of concurrent modifications. Sequential enrichment by this method therefore targets novel proteoforms for further investigation. With label-free quantification, we measured 3,353 oxidized Cys-sites from 1,457 proteins, where sequential phosphopeptide enrichment found 1,094 phosphosites on 720 redox proteins. This method uniquely builds on our knowledge of proteoforms in photosynthetic organisms, thereby identifying potentially novel control mechanisms of cellular machinery.