A Proteome Strategy for Fractionating Proteins and Peptides Using Continuous Free-Flow Electrophoresis Coupled Off-Line to Reversed-Phase High-Performance Liquid Chromatography

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• 作者：Robert L. Moritz ; Hong Ji ; Fré ; dé ; ric Schü ; tz ; Lisa M. Connolly ; Eugene A. Kapp ; Terence P. Speed ; and Richard J. Simpson
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：August 15, 2004
• 年：2004
• 卷：76
• 期：16
• 页码：4811 - 4824
• 全文大小：656K
• 年卷期：v.76,no.16(August 15, 2004)
• ISSN：1520-6882

文摘

Extensive prefractionation is now considered to be anecessary prerequisite for the comprehensive analysis ofcomplex proteomes where the dynamic range of proteinabundances can vary from ~10⁶ for cells to ~10¹⁰ fortissues such as blood. Here, we describe a high-resolution2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complexprotein mixtures by solution-phase isoelectric focusing(IEF) into 96 well-defined pools, each separated by~0.02-0.10 pH unit depending on the gradient created,followed by rapid (~6 min per analysis) reversed-phasehigh-performance liquid chromatography (RP-HPLC) ofeach FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plotsprotein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the seconddimension. By coupling a diode-array detector in line witha multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by bothUV absorbance and intrinsic fluorescence simultaneouslyfrom a single experiment. Triplicate analyses of standardproteins using a pH 3-10 gradient conducted over a3-day period revealed a high system reproducibility witha SD of 0.57 (0.05 pH unit) within the FFE pools and0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstratethat the FFE-IEF/RP-HPLC separation strategy can alsobe applied to complex mixtures of low molecular weightcompounds such as peptides. With the facile ability tomeasure the pH of the isoelectric focused pools, peptidepI values can be estimated and used to qualify peptideidentifications made using either MS/MS sequencingapproaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-basedFFE-IEF/RP-HPLC system is 6720.