title: Enrichment of phosphopeptides using biphasic immobilized metal affinity-reversed phase microcolumns.
abstract: Immobilized metal affinity chromatography (IMAC) based on Fe (3+) or Ga (3+) chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of beta-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of beta-casein monophosphorylated peptide in 20 microL of a solution of human serum protein digest (from 200 microg total serum protein after digestion and desalting).
authors.affiliation: Department of Pharmacology and MUSC Proteomics Center, Medical University of South Carolina, Charleston, SC 29425, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Schilling
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Knapp
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001798:Blood Proteins; D008670:Metals; D010748:Phosphopeptides