title: Detection and quantitation of SUMO chains by mass spectrometry.
abstract: The small ubiquitin-like modifiers (SUMOs) alter the function of cellular proteins by covalent attachment to lysine side-chains. SUMOs can target themselves for modification so generating SUMO polymers, the functions of which are beginning to be unraveled.The identification and quantitation of SUMO chains is essential for the functional investigation of SUMO polymerization. Classical techniques, such as site-directed mutagenesis and western blotting, are indirect and often inconclusive methods for the study of SUMO polymers. On the contrary, direct detection is possible with mass spectrometry (MS) by the identification of the SUMO-SUMO branched peptide remnant after proteolytic digestion. In this chapter, we describe a straightforward workflow that incorporates a modified database to efficiently detect SUMO polymers from simple and complex protein samples. In combination with stable isotope labeling by amino acids in cell culture (SILAC), this proteomic strategy allows accurate relative quantitation of SUMO polymers from different biological samples.
authors.affiliation: Wellcome Trust Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, Scotland, UK. i.matic@dundee.ac.uk
authors.forename: Ivan
authors.initials: I
authors.lastname: Matic
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D030562:Databases, Protein; D001330:Electronic Data Processing; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis* / Q000737:chemistry*
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins