Novel Mass Spectrometry-based Method Development and Applications to Signaling Peptides and Proteins

This dissertation presents research centered on mass spectrometry (MS)-based methods for peptide and protein analysis. All investigations belong to one of two groups: innovative application of well-established MS approaches to signal transduction systems, or development of novel peptide and protein analytical methods that could be applied to signaling peptides and proteins in the future. In Chapter 1, the overall goals and key findings of each project are summarized. In Chapter 2, the use of ion mobility (IM)-MS to characterize cis/trans isomers of proline-containing peptides was given a critical examination. As a result, the investigation yielded the first concrete example of experimental IM-MS evidence suggesting cis/trans isomers while theoretical evidence suggested exclusively trans-proline conformations. In Chapter 3, the analytical utility of IM-MS was used to mitigate isobaric interference that arises during the use of quantitative tandem mass tags. When co-eluting, co-isolated labeled peptides are fragmented, they yield chimeric reporter ion spectra. If the precursors could be separated by IM prior to fragmentation, accurate reporter intensities were obtained. Chapter 4 demonstrates the synergistic combination of high-throughput bottom-up proteomics and ion collision cross-section (CCS) measurement via IM-MS. After developing and validating a new method, it was used to create a peptide CCS database containing several thousand entries. Chapter 5 departs from IM-MS and method development to discuss the current state of proteomics in the field of chronic pain research. Chapter 6 then describes an application of multiplexed quantitative proteomics to the spared-nerve injury model of neuropathic pain. Among many proteins that were identified as having significant differential regulation, Bag3 showed the highest up-regulation in the pain model specimens and was orthogonally validated by Western blot. Chapter 7 reports on the application of multiplex quantitative proteomics to deciphering the complex signaling pathways of TGF-? and its role in restenosis. Representing the first biological application of dimethylated leucine tags to phosphoprotein analysis, several hundred phosphoproteoforms were identified as exhibiting significant differential regulation between normal smooth muscle cells and cells stimulated with TGF-?. Finally, Chapter 8 concludes the dissertation with an evaluation of the contributions made to the broader field of analytical chemistry.