Abstract
An online metal-free weak cation exchange-hydrophilic interaction LC/RPLC system has been developed for sensitive, high-throughput top-down MS. Here, we report results for analyzing PTMs of core histones, with a focus on histone H4, using this system. With just ∼24 μg on-column of core histones (H4, H2B, H2A, and H3) purified from human fibroblasts, 41 H4 isoforms were identified, with the type and location of PTMs unambiguously mapped for 20 of these variants. Compared to corresponding offline studies reported previously, the online weak cation exchange-hydrophilic interaction LC/RPLC platform offers significant improvement in sensitivity, with several orders of magnitude reduction in sample requirements and a reduction in the overall analysis time. To the best of our knowledge, this study represents the first online 2-D LC-MS/MS characterization of core histone mixture at the intact protein level.