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.