Separation and purification of multiply acetylated proteins using cation-exchange chromatography

Abstract

High-performance liquid chromatography (HPLC) is extremely useful for the study of proteins and the characterization of their posttranslational modifications. Here we describe a method that utilizes cation-exchange HPLC to separate multiply acetylated histone H3 species on the basis of their charge and hydrophilicity. This high-resolution method allows for the separation of histone H3 species that differ by as few as one acetyl group, and is compatible with subsequent analysis by a variety of techniques, including mass spectrometry and western blotting.

Fig. 1

In vitro acetylation of proteins using acetic anhydride. (a) Unmodified, recombinant histone H3 was treated with acetic anhydride (Ac₂O) and analyzed by dot blot immunodetection using antibodies specific to acetylated histone H3 or total H3. (b) The extent of in vitro acetylation of Tetrahymena histone H3 was measured by acid–urea gels, which combine size- and charge-based protein separation. The untreated H3 sample contains six bands with each band containing the indicated number of acetyl marks. Treatment with acetic anhydride generates hyperacetylated H3, which is represented by a significant upward shift in acid–urea gels.

Fig. 2

Flowchart of mobile-phase preparation for PolyCAT A chromatography. Circulating arrows indicate that stirring is required at that step.

Fig. 3

Separation of acetylated proteins using polyCAT A chromatography. (a) Dot blot analysis comparing Tetrahymena H3 species that were either hyperacetylated in vitro using Ac₂O or endogenously acetylated. These samples represent the extremes of acetylation that may exist in a biologically relevant sample and serve as ideal starting material to optimize polyCAT A chromatography conditions. (b) Chromatogram of an optimized polyCAT A run using 75 μg of endogenously acetylated Tetrahymena histone H3. Solutions described in Fig. 2 were used with the following chromatography protocol at a rate of 0.4 mL/min: inject sample; 0% B for 10 min; 0–10% B for 10 min; 10% B for 15 min; 10–50% B for 65 min; 50–100% B for 10 min; 100% B for 10 min; 100–0% B for 5 min; and 0% B for 10 min. Sub-peaks within each major acetylation peak likely represent differently methylated H3 species. (c) Peaks 0–4 from (b) were desalted and analyzed by acid-urea electrophoresis and Coomassie staining.