Mass Spectrometry Methods for the Analysis of Isopeptides Generated from Mammalian Protein Ubiquitination and SUMOylation

Navin Chicooree and Duncan L. Smith

Biological Mass Spectrometry, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, UK

Overview of Ub and SUMO

Biological Overview of Ubiquitin-Like Proteins

In addition to the ubiquitin (Ub) protein, ubiquitin-like proteins (Ubls) are a related super family of proteins that are involved in the post-translational modification (PTM) of target proteins. There are at least 14 Ubls including; small ubiquitin-like modifier (SUMO) proteins that have been identified in all systems spanning from prokaryotes to mammals. With the exception of the Pups, they share a similar three-dimensional core structure but are significantly different in their primary structure [1]. The main focus of this chapter is to review recent developments in applicable LC-MS/MS methods that improve bottom-up proteomic analyses of isopeptides derived from ubiquitin- and SUMO-modified proteins.

Activated forms of these two types of PTMs predominantly modify proteins through a mechanism of attachment by forming a covalent isopeptide bond between e-amino groups of lysine residues on proteins targeted for PTM. Although it occurs significantly less frequently, ubiquitination can also occur through a peptide covalent bond between the C-termini of activated Ub/Ubls and N-terminal a-amino groups of proteins [2]. In addition, ubiquitination has been reported to occur by covalent attachment at the hydroxyl group of target acceptor threonine and serine residues or through the thiol group of target acceptor cysteine residues [3, 4]. Similarly, SUMOylation has been reported to predominantly occur via the isopeptide bond of e-amino group of the target acceptor lysine. This chapter focuses on methods enabling the analysis of lysine ubiquitination and SUMOylation, which has occurred via the isopeptide bond of e-amino group of the target acceptor lysine.

Analysis of Protein Post-Translational Modifications by Mass Spectrometry,

First Edition. Edited by John R. Griffiths and Richard D. Unwin.

© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

< Prev   CONTENTS   Source   Next >