Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion.

Journal of Mass Spectrometry : JMS
Omoruyi OsulaRobert J Cotter

Abstract

Small-ubiquitin-like modifier (SUMO) is a posttranslational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization and mitosis, among other cellular processes, are known to occur. However, although the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly "tag" that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32-amino-acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approa...Continue Reading

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Citations

Apr 3, 2013·Analytical Biochemistry·Pawel P OlszowyPawel S Ciborowski
Aug 15, 2013·Rapid Communications in Mass Spectrometry : RCM·Navin ChicooreeDuncan L Smith
Jul 18, 2015·The FEBS Journal·Karolin Eifler, Alfred C O Vertegaal
Dec 6, 2014·Journal of Mass Spectrometry : JMS·Amanda E LeeCatherine Fenselau
Jan 24, 2013·Current Opinion in Chemical Biology·Benedikt M Kessler
Jun 8, 2017·Chemical Communications : Chem Comm·Yan ZhangChun-Yang Zhang
Jul 28, 2020·Current Protein & Peptide Science·Madhuri JayathirthaCostel C Darie

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