Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome

Research output: ThesisDoctoral thesis 1 (Research UU / Graduation UU)

Abstract

The work, described in this thesis, has been aimed at the improvement of proteomic workflows; from the sample preparation, to the optimization of state-of-art chromatographical separations and alternative MS fragmentation techniques. An important focus of this work is represented by the analysis of peptides harboring various post translational modifications (PTMs) such as glycosylation, phosphorylation, methylation and di-methylation. PTM modified peptides often require customized approaches in order to improve their detection. Furthermore advancements in LC-MS/MS approaches can enhance the detection of endogenous peptides eluted from HLA class I and II molecules, which present exotic characteristics when compared to ‘standard’ tryptic peptides obtained in proteomic workflows. First we discuss the development and implementation of an online 2D-dimensional SCX/RP UHPLC system coupled to a fast sequencing and high resolution MS. In this work we asked the question whether the fast and sensitive 2D LC-MS/MS workflow could compete with a 1D, in terms of proteome coverage, analysis time and sample usage. Next we explore a method based on several stages of state-of-art chromatographical techniques, high resolution MS and alternative fragmentation to define kinase consensus motifs with high accuracy which can be implemented for any kinase. We further demonstrate that recent developments in MS-based sequencing technology can expand the detectable peptide repertoire revealing unique features in the antigen presentation machinery.By analyzing a vast repertoires of HLA class I eluted peptides, we found that they can harbor, little explored PTMs such as arginine methylation and di-methylation. Interestingly, we also found evidences of unusual glycosylated HLA class I-bound peptides. The observed O-linked glycans are extended O-GlcNAc rather than GalNAc-initiated O-glycans. In fact O-GlcNAc on an HLA peptide may present a special case because of the HLA class I pathway traffics through the ER and Golgi on its way to the cell membrane, and can hence be exposed to glycosyltransferases that further modify the initiator O-GlcNAc group. In another work on PTMs harboring HLA class I peptides, we charted the phosphopeptides bound to four distinct HLA-B molecules and observed that they shared a preference for peptides phosphorylated at position 4. We investigated the structural basis for this observation concluding that the observed phosphorylation motif may be common to most HLA-B molecules.Alterations in PTMs are a recognized hallmark of diseases, therefore these kind of PTMs can potentially provide a unique source of HLA class I-presented peptide antigens that could uniquely stimulate the immune response. Moreover, the knowledge added by our works provides a base for the improved prediction and identification of PTMs neo-antigens, as potentially used for cancer immunotherapy. Lastly, we evaluate a mass MS-based analysis of HLA-DR-associated self-peptides. The complementary peptide fragmentation techniques ETD, EThcD and HCD were employed to improve the identification and coverage of HLA-DR-associated peptides. The data suggest selection of proteins in the HLA class II antigen processing and presentation pathway, related to the route of antigen delivery, diversity in proteolytic activity and strict selection by HLA-DR molecules.
Original languageEnglish
Awarding Institution
  • Utrecht University
Supervisors/Advisors
  • Heck, Albert, Primary supervisor
  • Mohammed, S., Co-supervisor
Award date29 Aug 2016
Publisher
Publication statusPublished - 29 Aug 2016

Keywords

  • Alternative fragmentations
  • HLA class I
  • HLA class II
  • phosphorylation
  • methylation
  • glycosylation

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