A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography
Publication Year 2004-08-15, Volume 76, Issue #16, Page 4811-4824
- Journal Title
- ANALYTICAL CHEMISTRY
- Publication Type
- Journal Article
- Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from similar to10(6) for cells to similar to10(10) for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by similar to0.02-0.10 pH unit depending on the gradient created, followed by rapid (similar to6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydro-phobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.
- AMER CHEMICAL SOC
- IMMOBILIZED PH GRADIENTS; ASSISTED-LASER-DESORPTION/IONIZATION; ELECTRON-CAPTURE DISSOCIATION; MASS-SPECTROMETRIC ANALYSIS; AMINO-ACID-SEQUENCES; 2-DIMENSIONAL ELECTROPHORESIS; SAMPLE PREPARATION; IDENTIFICATION TECHNOLOGY; MEMBRANE-PROTEINS; ACCURATE MASS
- Publisher's Version
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Creation Date: 2004-08-15 12:00:00Last Modified: 0001-01-01 12:00:00