New highly conserved ubiquitin phospho-Ser65 peptide is up-regulated upon cell treatment with CCCP. Flp-In T-Rex HEK-293 cells stably expressing FLAG-empty, wild-type PINK1–FLAG or kinase-inactive PINK1–FLAG were grown in light, heavy and medium SILAC media respectively. Cells under each condition were stimulated with 10 μM CCCP for 3 h. Subsequently, membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest. Lysates from each of the three conditions were mixed at 1:1:1 and digested with trypsin before phosphopeptide enrichment by HILIC (hydrophilic-interaction LC) and TiO2, and analysis by MS. Data analysis was performed using MaxQuant. The experiment was performed using four replicates. (A) Representative extracted ion chromatograms representing the ubiquitin Ser65) phosphopeptide TLSDYNIQKEpSTLHLVLR in the three SILAC-labelled conditions. (B) Sequence alignment of residues around Ser65 in human Parkin and ubiquitin in a variety of organisms showing a high degree of conservation. C. elegans, Caenorhabditis elegans; D. melanogaster, Drosophila melanogaster; H. sapiens, Homo sapiens; M. musculus, Mus musculus; S. cerevisiae, Saccharomyces cerevisiae.
