title: Dephosphorylation of cardiac proteins in vitro - a matter of phosphatase specificity.
abstract: Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the extent of this modification in nature. To date there is still a lack of information about phosphatase specificity for different proteomes and their conditions to achieve maximum enzyme activity. This information is important per se, and in addition often requested in functional and biochemical in vitro studies, where a dephosphorylated sample is needed as a negative control to define baseline conditions. In this study, we have addressed the effectiveness of two phosphatases endogenously present in the heart (protein phosphatases 1 and 2A) and two generic phosphatases (alkaline phosphatase and lambda protein phosphatase) on three cardiac subproteomes known to be regulated by phosphorylation. We optimized the dephoshorylating conditions on a cardiac tissue fraction comprising cytosolic and myofilament proteins using 2DE and MS. The two most efficient conditions were further investigated on a mitochondrial-enriched fraction. Dephosphorylation of specific proteins depends on the phosphatase, its concentration, as well as sample preparation including buffer composition. Finally, we analyzed the efficiency of alkaline phosphatase, the phosphatase with the broadest substrate specificity, using TiO(2)  peptide enrichment and 2DLC-MS/MS. Under these conditions, 95% of the detected cardiac cytoplasmic-enriched phospho-proteome was dephosphorylated. In summary, targeting dephosphorylation of the cardiac muscle subproteomes or a specific protein will drive the selection of the specific phosphatase, and each requires different conditions for optimal performance.
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Cathrine
authors.initials: C
authors.lastname: Husberg
authors.forename: Giulio
authors.initials: G
authors.lastname: Agnetti
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Holewinski
authors.forename: Geir
authors.initials: G
authors.lastname: Christensen
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
mesh_terms: D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D009124:Muscle Proteins; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2