"ChopNSpice," a mass spectrometric approach that allows identification of endogenous small ubiquitin-like modifier-conjugated peptides.
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.
Hsiao
He-Hsuan
HH
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Meulmeester
Erik
E
Frank
Benedikt T C
BT
Melchior
Frauke
F
Urlaub
Henning
H
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D012984:Software
D000906:Antibodies; D010455:Peptides; D025841:Small Ubiquitin-Related Modifier Proteins