title: A proteomic strategy for gaining insights into protein sumoylation in yeast.
abstract: Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction, and cell division. However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood. In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass. Specifically, SUMO-conjugated proteins were isolated by a double-affinity purification procedure from a Saccharomyces cerevisiae strain engineered to express tagged SUMO. The components of the isolated protein mixture were then identified by subsequent LC-MS/MS analysis using an LTQ FT mass spectrometer. In this manner, 159 candidate sumoylated proteins were identified by two or more peptides. Furthermore, the high accuracy of the instrument, combined with stringent search criteria, enabled the identification of an additional 92 putative candidates by only one peptide. The validity of this proteomics approach was confirmed by performing subsequent Western blot experiments for numerous proteins and determining the actual sumoylation sites for several other substrates. These data combine with recent works to further our understanding of the breadth and impact of protein sumoylation in a diverse array of biological processes.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Carilee
authors.initials: C
authors.lastname: Denison
authors.forename: Adam D
authors.initials: AD
authors.lastname: Rudner
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gerber
authors.forename: Corey E
authors.initials: CE
authors.lastname: Bakalarski
authors.forename: Danesh
authors.initials: D
authors.lastname: Moazed
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins