Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

被引：49

作者：

Hata, K

Morisaka, H

Hara, K

Mima, J

Yumoto, N

Tatsu, Y

Furuno, M

Ishizuka, N

Ueda, M ^{[1
]}

机构：

[1] Kyoto Univ, Grad Sch Agr, Div Appl Life Sci, Sakyo Ku, Kyoto 6068502, Japan

[2] Natl Inst Adv Ind Sci & Technol, Osaka 5638577, Japan

[3] GL Sci Inc, Iruma, Saitama 3580032, Japan

[4] Emaus Kyoto Inc, Ukyo Ku, Kyoto 6150055, Japan

来源：

ANALYTICAL BIOCHEMISTRY | 2006年 / 350卷 / 02期

关键词：

phosphoproteome; 2D HPLC; Titania column; monolithic silica column;

D O I：

10.1016/j.ab.2006.01.009

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 P-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. (c) 2006 Elsevier Inc. All rights reserved.

引用

页码：292 / 297

页数：6

共 15 条

[1] Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry [J].