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

Abstract

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 approximately 10(6) for cells to approximately 10(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 approximately 0.02-0.10 pH unit depending on the gradient created, followed by rapid (approximately 6 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 hydrophobicity (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.

Proteome Analysis of Microdissected Formalin-fixed and Paraffin-embedded Tissue Specimens

“…For profiling tryptic peptides obtained from an FFPE tissue sample, the entire content of focused peptides in the CIEF capillary was split into 19 individual fractions (Figure 2), which were further resolved by nano-RPLC and identified using nano-ESI-LTQ-MS/MS. The number of distinct peptide identifications measured from each CIEF fraction is significantly greater than that typically reported in the literature using other IEF techniques, including immobilized pH gradient gels (Cargile et al 2004;Essader et al 2005) and gel-free approaches (Wall et al 2000;Zuo et al 2001;Yan et al 2003;Moritz et al 2004) such as chromatofocusing, immobilized pH membranes, Rotofor, and free-flow electrophoresis. A key feature of our CIEF-based multidimensional separation technology is the elimination of protein/peptide loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein expression profiles within FFPE tissue specimens.…”

“…A key feature of our CIEF-based multidimensional separation technology is the elimination of protein/peptide loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein expression profiles within FFPE tissue specimens. By contrast, preparative-scale IEF techniques (Wall et al 2000;Zuo et al 2001;Yan et al 2003;Cargile et al 2004;Moritz et al 2004;Essader et al 2005) are incompatible with the smaller scale of the more selectively procured proteomes obtained from microdissection-procured tissues.…”

Interpretation of Shotgun Proteomic Data

“…However, gel-based methods have known limitations such as low detection sensitivity, bias toward high abundance proteins, and difficulty in resolving internal membrane or basic proteins. Non-gel-based multidimensional protein separation methods are being developed and can circumvent some of these limitations (101)(102)(103)(104)). Another promising top-down protein characterization technique is based on MS/MS sequencing of intact proteins (5,6,105).…”

The use of proteomics to identify novel therapeutic targets for the treatment of disease

“…This method can be used to maintain the native conformation of proteins, but several proteins might resolve in a single 1D band making specific protein identification difficult. However, this technology has additional benefits as it can also be combined with chromatographic separation methods or direct analysis by mass spectrometry thereby completely avoiding any gel-based protein separation (Wang et al 2003;Harper et al 2004;Moritz et al 2004;Xiao et al 2004).…”