Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns |
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Authors: | Hata Koji Morisaka Hironobu Hara Keisuke Mima Joji Yumoto Noboru Tatsu Yoshiro Furuno Masahiro Ishizuka Norio Ueda Mitsuyoshi |
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Affiliation: | Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. |
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Abstract: | Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses. |
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Keywords: | Phosphoproteome 2D HPLC Titania column Monolithic silica column |
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