title: Determining the Mitochondrial Methyl Proteome in Saccharomyces cerevisiae using Heavy Methyl SILAC.
abstract: Methylation is a common and abundant post-translational modification. High-throughput proteomic investigations have reported many methylation sites from complex mixtures of proteins. The lack of consistency between parallel studies, resulting from both false positives and missed identifications, suggests problems with both over-reporting and under-reporting methylation sites. However, isotope labeling can be used effectively to address the issue of false-positives, and fractionation of proteins can increase the probability of identifying methylation sites in lower abundance. Here we have adapted heavy methyl SILAC to analyze fractions of the budding yeast Saccharomyces cerevisiae under respiratory conditions to allow for the production of mitochondria, an organelle whose proteins are often overlooked in larger methyl proteome studies. We have found 12 methylation sites on 11 mitochondrial proteins as well as an additional 14 methylation sites on 9 proteins that are nonmitochondrial. Of these methylation sites, 20 sites have not been previously reported. This study represents the first characterization of the yeast mitochondrial methyl proteome and the second proteomic investigation of global mitochondrial methylation to date in any organism.
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute and ‡Department of Biological Chemistry and the David Geffen School of Medicine, UCLA , Los Angeles, California 90095, United States.
authors.forename: Katelyn E
authors.initials: KE
authors.lastname: Caslavka Zempel
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute and ‡Department of Biological Chemistry and the David Geffen School of Medicine, UCLA , Los Angeles, California 90095, United States.
authors.forename: Ajay A
authors.initials: AA
authors.lastname: Vashisht
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute and ‡Department of Biological Chemistry and the David Geffen School of Medicine, UCLA , Los Angeles, California 90095, United States.
authors.forename: William D
authors.initials: WD
authors.lastname: Barshop
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute and ‡Department of Biological Chemistry and the David Geffen School of Medicine, UCLA , Los Angeles, California 90095, United States.
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute and ‡Department of Biological Chemistry and the David Geffen School of Medicine, UCLA , Los Angeles, California 90095, United States.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Clarke
mesh_terms: D007553:Isotope Labeling; D008745:Methylation; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D024101:Mitochondrial Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins
keywords: MudPIT; heavy methyl SILAC; methyl proteome; methylation; mitochondria; protein arginine methylation; protein lysine methylation; yeast