title: A rapid screening assay to search for phosphorylated proteins in tissue extracts.
abstract: Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO(2) enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.
authors.affiliation: Centre for Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.
authors.forename: Ignazio
authors.initials: I
authors.lastname: Garaguso
authors.forename: Juergen
authors.initials: J
authors.lastname: Borlak
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D019295:Computational Biology / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008168:Lung / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010447:Peptide Hydrolases / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014020:Tissue Extracts / Q000737:chemistry*
chemical_list: D010750:Phosphoproteins; D014020:Tissue Extracts; D010447:Peptide Hydrolases