Mass Spectrometric Analyses of Post-Translationally Modified Proteins

Abstract

Protein post-translational modifications (PTMs) possess key functions in the regulation of various cellular processes. In this thesis, several new technologies are developed to map protein PTMs, containing phosphorylation, glycosylation and SUMOylation. Five major topics are presented in this thesis. In Chapter 1- General Introduction, describes what is proteomics, the importance of protein PTMs, the main techniques in proteomic analysis, including the principle of technologies for protein and peptide separation, the theory of mass spectrometry (MS) and concept of proteomic data analysis. In Chapter 2 - A High-Throughput Method for Phosphopeptide Enrichment of Spliceosomal Proteins and Its Application, a disposable TiO2 microspin column is fabricated in-house for enrichment of phosphopeptides. The method offers several advantages, including high-throughput, easy to use, low cost, high selectivity and sensitivity. In combination with different proteomic strategies, 1381 unique phosphorylation sites corresponding to 390 distinct proteins were identified in spliceosomal proteins. We further applied this method to explore the phosphorylation sites on PRP6, PRP31 and SLP65 and to study protein-RNA interaction by UV-induced crosslinking reaction. In Chapter 3 - Efficient Enrichment of Intact Phosphoproteins prior to Mass Spectrometric Analysis, a straightforward and reliable phosphoprotein purification procedure was developed based on calcium phosphate precipitation (CPP). Integration of TiO2 microspin column, a total of 192 unique phosphorylation sites corresponding to 45 distinct proteins were identified from the U1 small nuclear ribonucleoproteins (snRNPs); of these, 59 phosphorylation sites were not reported previously. In Chapter 4 - Pseudo-Neutral-Loss Scan for Selective Detection of Phosphopeptides and N-Glycopeptides using Liquid Chromatography Coupled with a Hybrid Linear Ion-Trap / Orbitrap Mass Spectrometer, a pseudo-neutral-loss scan on a hybrid LTQ-Orbitrap MS was built up for selectively probing phosphopeptides and glycopeptides. The presence of known characteristic mass pair (phosphoric acid or monosaccharide residues) in the spectrum during in-source collision-induced dissociation (CID) was selected to trigger MS/MS and multi-stage activation MS3 fragmentation. Our method is compatible with nano-liquid chromatography (nano-LC) for separation of complex peptide mixtures without any further enrichment. The consequent spectra provide peptide sequence identification and modified site assignment as well as information of the glycan structure. In Chapter 5 - ChopNSpice , a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides, a novel, user-friendly and straightforward database search tool was developed, called ChopNSpice , to unambiguously determine the mammalian SUMO1 and SUMO2/3 conjugation sites in vitro and in vivo by mass spectrometry in combination with MS-based search engines like MASCOT or Sequest. High mass data dependent acquisition (DDA) is highly suitable for the accurate detection and sequencing of larger peptides and additionally facilitates detection of lower abundant SUMO-conjugates. We demonstrated the power of ChopNSpice software in combination with proteomic strategy, resulting in the identification of 10 SUMOylated proteins corresponding to 17 distinct sites in endogenous Hela-S3 cells. 15 SUMOylated sites were identified in this study appeared to be novel, which may provide a valuable resource to the biological research community.