Multiplexed size separation of intact proteins in solution phase for mass spectrometry

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.