title: Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis.
abstract: We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Mazanek
authors.forename: Goran
authors.initials: G
authors.lastname: Mituloviae
authors.forename: Franz
authors.initials: F
authors.lastname: Herzog
authors.forename: Christoph
authors.initials: C
authors.lastname: Stingl
authors.forename: James R A
authors.initials: JR
authors.lastname: Hutchins
authors.forename: Jan-Michael
authors.initials: JM
authors.lastname: Peters
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
chemical_list: D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium