Evaluation of Comprehensive Multidimensional Separations Using Reversed-Phase, Reversed-Phase Liquid Chromatography/Mass Spectrometry for Shotgun Proteomics

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• 作者：Tatsuji Nakamura ; Junro Kuromitsu ; Yoshiya Oda
• 刊名：Journal of Proteome Research
• 出版年：2008
• 出版时间：March 2008
• 年：2008
• 卷：7
• 期：3
• 页码：1007 - 1011
• 全文大小：1200K
• 年卷期：v.7,no.3(March 2008)
• ISSN：1535-3907

文摘

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.