Mamalian protein ubiquitination and SUMOylation are reversible post translational modifications, which are involved in a multitude of important complex regulatory processes within the cell. Current mass spectrometry approaches that involve bottom-up proteomics to comprehensively analyse these modifications, have proved to be problematic. In this work, analytical approaches are carried out to improve and enhance the comprehensive analysis of these modifications. Tryptic proteolysis of ubiquitinated proteins results in the generation of isopeptides bearing adi-glycine (GG) remnant. Current mass spectrometry approaches used to identify these isopeptides are predominantly reliant on detecting the signature mass shift of the GG remnant (114.043 Da). The lack of sequence information from the GG remnant post MS/MS acquisition results in database search algorithms falsely identifiying these isopeptides. Reductive methylation chemistry was employed to derivatize these isopeptides. Upon collision induced dissociation of the isopeptides two robust ions were released from the iso-N-terminus of the GG remnant ; i) an a1’ ion at m/z 62.09, corresponding to the G of the remnant and ii) a b2’ ion at m/z 147.11, corresponding to the full GG remnant. Post-acquisition data extraction of these unique diagnostic ions demonstrated enhanced selectivity towards identifying these isopeptides. Tryptic proteolysis of SUMOylated proteins results in the generation of isopeptides bearing a substantial iso-C-terminal SUMO remnant. The CRA(K) (Consecutive Residue Addition tolysines (K)) approach combined independant use of proteolytic enzymes and unbiased consecutive residue addition of amino acids pertaining to these iso-C-terminal SUMOremnants, on all lysine residues. This approach enabled the identification of analytically useful novel wildtype isopeptides derived from the proteolysis of SUMO(1/2/3)ylated proteins, bearing GG, TGG and QTGG remnants. The analytically useful isopeptides derived from proteolysis of SUMO(2/3)ylated proteins lacked robust diagnostic information from their iso-C-terminal bearing TGG and QTGG remnants. Reductive methylation chemistry was utilised to derivatize these isopeptides and enabled diagnostic a’ and b’ ions to be released from their iso-N-termini; i) a1’ (m/z 133.13),b2’ (m/z 262.17) and b4’ (m/z 376.22) ions, corresponding to the QTGG remnant and ii) (m/z106.10), b2’ (m/z 191.14) and b3’ (m/z 248.14) ions, corresponding to the TGG remnant. Post-acquisition data extraction of these unique diagnostic ions, enabled comprehensive structural elucidation of these isopeptides and enhanced selectivity towards identification.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:632209 |
| Date | January 2014 |
| Creators | Chicooree, Navin |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/enhancing-the-mass-spectrometric-analysis-of-ubiquitinlike-modifications(ab42ea7c-73d1-4348-85e5-69813be451f7).html |
Page generated in 0.002 seconds