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Proteome-wide identification of SUMO modification sites by mass spectrometry.

Abstract：

:The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific consequences of SUMO modification requires knowledge of the location of conjugation sites, and we have developed a straightforward protocol for the proteome-wide identification of SUMO modification sites using mass spectrometry (MS). The approach described herein requires the expression of a mutant form of SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) is replaced with a lysine (SUMO(KGG)). Digestion of SUMO(KGG) protein conjugates with endoproteinase Lys-C yields a diGly motif attached to target lysines. Peptides containing this adduct are enriched using a diGly-Lys (K-ɛ-GG)-specific antibody and identified by MS. This diGly signature is characteristic of SUMO(KGG) conjugation alone, as no other ubiquitin-like protein (Ubl) yields this adduct upon Lys-C digestion. We have demonstrated the utility of the approach in SUMOylation studies, but, in principle, it may be adapted for the site-specific identification of proteins modified by any Ubl. Starting from cell lysis, this protocol can be completed in ∼5 d.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Tammsalu T,Matic I,Jaffray EG,Ibrahim AF,Tatham MH,Hay RT

doi

10.1038/nprot.2015.095

subject

Has Abstract

pub_date

2015-09-01 00:00:00

pages

1374-88

issue

9

eissn

1754-2189

issn

1750-2799

pii

nprot.2015.095

journal_volume

10

pub_type

杂志文章

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