Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis.

SDS has recognized benefits for protein sample preparation, including solubilization and imparting molecular weight separation (e.g., SDS-PAGE). Here, we compare two proteome workflows which incorporate SDS for protein separation, namely, SDS-PAGE coupled to LC/MS (GeLC MS), along with a solution separation platform, GELFrEE, for intact proteome prefractionation and identification. Despite the clear importance of SDS in these and other proteome analysis workflows, the affect of SDS on an LC/MS proteome experiment has not been quantified. We first examined the influence of SDS on both a bottom-up as well as a top-down (intact protein) MS workflow. Surprisingly, at levels up to 0.01% SDS in the injected sample, reliable MS characterization is obtained. We subsequently explored organic precipitation protocols (chloroform/methanol/water and acetone) as a means of lowering SDS, and present a simple modified acetone precipitation protocol which consistently enables MS proteome characterizations from samples initially containing 2% SDS. With this effective strategy for SDS reduction, the GELFrEE MS workflow for bottom-up proteome analysis was characterized relative to GeLC MS. Remarkable agreement in the number and type of identified proteins was obtained from these two separation platforms, validating the use of SDS in solution-phase proteome analysis.

Reliable size-based protein separation is an invaluable biological technique. Unfortunately, size separation in solution is underutilized, owing perhaps to the poor resolution of conventional techniques. Here, we report an enhanced multiplexed GELFrEE (gel-eluted liquid fraction entrapment electrophoresis) device which incorporates eight independent separation channels, operating with high repeatability. This enables simultaneous size separation of independent proteome samples, each into 16 well resolved liquid fractions, covering 10-150 kDa in 1.5 h. A novel strategy to increase sample loads while maintaining electrophoretic resolution is presented by distributing the sample among the eight channels with subsequent pooling of collected fractions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the S. cerevisiae proteome following GELFrEE separation and sodium dodecyl sulfate (SDS) removal demonstrates the resolution and high correlation achieved between molecular weight and fraction number for the identified proteins. This device is highly orthogonal to solution isoelectric focusing, enabling our disclosure of a fully multiplexed high-throughput two-dimensional liquid electrophoretic (2D LE) platform that separates analogously to 2D polyacrylamide gel electrophoresis (PAGE). With 2D LE, a total of 128 well-resolved liquid fractions are obtained from 1 mg of S. cerevisiae proteins covering ranges 3.8 < pI < 7.8 and 10 kDa < MW < 150 kDa in an unprecedented 3.25 h total separation time.

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A method for generating peptide mass maps from dilute protein samples is presented. The method involves the concentration of proteins from aqueous solution by adsorption onto reversed-phase polymeric microbeads. These beads are then washed extensively to remove contaminants, after which the bound proteins are digested with trypsin. Analysis of the digestion products is performed by MALDI-TOF mass spectrometry following direct deposition of the beads on a MALDI target, along with the matrix solution. The procedure is demonstrated using solutions of cytochrome c, lysozyme, and bovine serum albumin. The results of these digests are compared to trypsin digestions of the protein samples without sample preconcentration. Comparative results are also presented for protein solutions contaminated with 2 M NaCl, 2 M urea, or sodium dodecyl sulfate at concentrations up to 0.02%. These results reveal that, with the microbead preconcentration procedure, peptide mass maps can routinely be generated from highly contaminated samples with a protein concentration of only 100 nM.