Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

摘要

In this paper, we are evaluating the strategy of sortingnpeptides/proteins based on the charge to mass withoutnresorting to ampholytes and/or isoelectric focusing, usingna single- and two-step free-flow zone electrophoresis. Wendeveloped a simple fabrication method to create a saltnbridge for free-flow zone electrophoresis in PDMS chipsnby surface printing a hydrophobic layer on a glass substrate.nSince the surface-printed hydrophobic layer preventsnplasma bonding between the PDMS chip and thensubstrate, an electrical junction gap can be created fornfree-flow zone electrophoresis. With this device, we demonstratedna separation of positive and negative peptidesnand proteins at a given pH in standard buffer systems andnvalidated the sorting result with LC/MS. Furthermore, wencoupled two sorting steps via off-chip titration and isolatednpeptides within specific pI ranges from sample mixtures,nwhere the pI range was simply set by the pH values ofnthe buffer solutions. This free-flow zone electrophoresisnsorting device, with its simplicity of fabrication, and ansorting resolution of 0.5 pH unit, can potentially be a highthroughputnsample fractionation tool for targeted proteomicsnusing LC/MS.