Study of Peroxisomal Protein Phosphorylation by Functional Proteomics.

Reversible protein phosphorylation is a frequently occurring posttranslational modification mediated by protein kinases and phosphatases that plays an essential role in the regulation of a large number of cellular processes. Evidence is accumulating that protein phosphorylation is also an important mechanism governing processes associated with peroxisome biology. For an improved and detailed understanding of these processes and their regulation it is therefore crucial to study phosphorylation of peroxisome-associated proteins and to determine the phosphorylated amino acid(s). To place peroxisome-related processes into a larger, cellular context, it is further required to identify the kinases and phosphatases catalyzing phosphorylation and dephosphorylation events in peroxisomal proteins. We here provide a strategy for the targeted analysis of peroxisomal phosphoproteins of Saccharomyces cerevisiae combining affinity purification of epitope-tagged peroxisomal proteins with Phos-tag SDS-PAGE and high-resolution mass spectrometry (MS) for the identification and precise localization of in vivo phosphosites. Furthermore, we describe a protocol for an MS-based in vitro kinase assay using recombinant peroxisomal proteins and a selected kinase facilitating the site-resolved analysis of kinase-substrate relationships.