Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn

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• 作者：Masaki Wakabayashi ; Yutaka Kyono ; Naoyuki Sugiyama ; Yasushi Ishihama
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：October 20, 2015
• 年：2015
• 卷：87
• 期：20
• 页码：10213-10221
• 全文大小：497K
• ISSN：1520-6882

文摘

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鈥?00 nonredundant phosphopeptides from triplicate analyses of 100 渭g 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.