Abstract

High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.