Mass Spectrometry Applications for Comparative Proteomics and Peptidomic Discovery.

摘要

In this thesis, multiple biological samples from various diseases models or treatments are investigated using shotgun proteomics and improved methods are developed to enable extended characterization and detection of neuropeptides. In general, this thesis aims to expand upon the rapidly evolving field of mass spectrometry (MS)-based proteomics and peptidomics by primarily enhancing small scale sample analysis. A review of the current status and progress in the field of biomarker discovery in peptidomics and proteomics is presented. To this rapidly expanding body of literature, our critical review offers new insights into MS-based biomarker studies investigating numerous biological samples, methods for post-translational modifications, quantitative proteomics, and biomarker validation. Methods are developed and presented, including immunodepletion for small volume cerebrospinal fluid (CSF) samples, for comparison of the CSF proteomes between an Alexander disease transgenic mouse model with overexpression of the glial fibrillary acidic protein and a control animal. This thesis also covers the application of the small scale immunodepletion of CSF for comparative proteomic analysis of a novel rat adapted scrapie (RAS) model for prion disease and compares the RAS CSF proteome to control rat CSF using MS. Large scale phosphoproteomics of starved vs. glucose fed yeast is presented to better understand the phosphoproteome changes that occur during glucose feeding. Method development for neuropeptide analysis is expanded upon using electron transfer dissociation (ETD) fragmentation to successfully sequence, for the first time, the crustacean hyperglycemic hormone precursor-related peptide (CPRP) from the blue crab Callinectes sapidus. In addition, a method for ETD sequencing of sulfonated neuropeptides using a magnesium salt adduct in an ion trap mass spectrometer is reported. This thesis also reports on a method for sub-µg peptide isolation when using a molecular weight cut-off filtration device to improve sample recovery by over 2 orders of magnitude. All the protocols used throughout the work are provided in an easy to use step-by-step format in the Appendix. Collectively, this body of work extends the capabilities of mass spectrometry as a bioanalytical tool for shotgun proteomics and expands upon methods for neuropeptide discovery and analysis.