Global identification of SUMO modification sites

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.<br/><br/>A method has been developed enabling global identification of sumoylated lysines by mass spectrometry (MS). The workflow entails <i>in vivo</i> conjugation to hexahistidine (6His)-tagged SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) has been mutated to lysine (6His-SUMO^KGG). Lys-C digestion of 6His-SUMO^KGG 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.<br/><br/>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.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2016

Author dc:creator

• Tammsalu, Triin

Advisor dc:contributor.advisor

• Hay, Ronald

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

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/5f8905a2-c31c-45ec-8173-4c91cf797342

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/5f8905a2-c31c-45ec-8173-4c91cf797342