title: Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.
abstract: In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool to study protein phosphorylation because it allows unbiased localization, and site-specific quantification, of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy to identify phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite the high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. Tandem metal-oxide affinity chromatography (MOAC) represents a robust and highly selective approach for the identification and site-specific quantification of low abundant phosphoproteins that is based on the successive enrichment of phosphoproteins and -peptides. This strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, tryptic digestion of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides. Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and, thus, allows probing of the phosphoproteome to unprecedented depth.
authors.affiliation: Department of Molecular Systems Biology, University of Vienna, Vienna, Austria.
authors.forename: Gerold J M
authors.initials: GJ
authors.lastname: Beckers
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hoehenwarter
authors.forename: Horst
authors.initials: H
authors.lastname: Röhrig
authors.forename: Uwe
authors.initials: U
authors.lastname: Conrath
authors.forename: Wolfram
authors.initials: W
authors.lastname: Weckwerth
mesh_terms: D000537:Aluminum Oxide / Q000737:chemistry*; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014025:Titanium / Q000737:chemistry*
chemical_list: D029681:Arabidopsis Proteins; D010750:Phosphoproteins; D020543:Proteome; C009495:titanium dioxide; D014025:Titanium; D000537:Aluminum Oxide