title: Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn.
abstract: We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO2) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 μg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , 46-29, Yoshida-Shimo-Adachi-Cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Wakabayashi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , 46-29, Yoshida-Shimo-Adachi-Cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kyono
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , 46-29, Yoshida-Shimo-Adachi-Cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Sugiyama
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , 46-29, Yoshida-Shimo-Adachi-Cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
mesh_terms: D002845:Chromatography / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D010766:Phosphorylation; D014025:Titanium / Q000737:chemistry*
chemical_list: D010455:Peptides; C009495:titanium dioxide; D014025:Titanium