Global identification of SUMO modification sites

  • Triin Tammsalu

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Posttranslational modification by Small Ubiquitin-like modifiers (SUMOs) affects the behaviour of proteins involved in diverse cellular processes and is indispensable for cell survival. Proteomic studies have identified hundreds of putative targets of SUMO conjugation, but understanding the substrate-specific consequences of the modification requires knowledge of precise target lysines. Procedures for the high-throughput mapping of SUMO modification sites, however, are lacking.

A method has been developed enabling global identification of sumoylated lysines by mass spectrometry (MS). The workflow entails in vivo conjugation to hexahistidine (6His)-tagged SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) has been mutated to lysine (6His-SUMOKGG). Lys-C digestion of 6His-SUMOKGG conjugates yields a diGly remnant on modified lysines that cannot be attributed to any other known modifier. This allows enrichment of SUMO remnant-modified peptides with a diGly-Lys (K-ε-GG)-specific antibody prior to their MS-based analysis.

Application of the workflow enabled the unambiguous identification of 612 SUMO2 conjugation sites from the nuclear fraction of unperturbed human cells and 8262 sites from the same fraction of heat-stressed cells. Exposure to proteotoxic stress increased the extent of substrate sumoylation, and targeted proteins with similar functions or found within the same macromolecular complexes. These data provide a comprehensive resource for future research on the physiological role of sumoylation in health and disease.
Date of Award2016
Original languageEnglish
Awarding Institution
  • University of Dundee
SponsorsWellcome Trust
SupervisorRonald Hay (Supervisor)

Keywords

  • SUMO
  • SUMO modification sites
  • Mass Spectrometry
  • Heat stress
  • Subcellular fractionation

Cite this

Global identification of SUMO modification sites
Tammsalu, T. (Author). 2016

Student thesis: Doctoral ThesisDoctor of Philosophy