“ChopNSpice,” a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides

• Published in: Molecular & cellular proteomics Vol. 8; no. 12; pp. 2664 - 2675
• Main Authors: Hsiao, He-Hsuan, Meulmeester, Erik, Frank, Benedikt T.C., Melchior, Frauke, Urlaub, Henning
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2009; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Antibodies - metabolism; Binding Sites; HeLa Cells; Humans; Immunoprecipitation; Mass Spectrometry - methods; Molecular Sequence Data; Molecular Weight; Peptides - analysis; Peptides - chemistry; Small Ubiquitin-Related Modifier Proteins - metabolism; Software
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M900087-MCP200

Conjugation of small ubiquitin-like modifier (SUMO) to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using MS. In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore, we developed a simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we identified SUMO acceptor sites in, among others, endogenous SUMO1, SUMO2, RanBP2, and Ubc9.