Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.

Top-down mass spectrometry (MS) analyzes intact proteins at the proteoform level, which allows researchers to better understand the functions of protein modifications. Recently, top-down proteomics has increased in popularity due to advancements in high-resolution mass spectrometers, increased efficiency in liquid chromatography (LC) separation, and advances in data analysis software. Some unique protein proteoforms, which have been distinguished using top-down MS, have even been shown to exhibit marked variation in biological function compared to similar proteoforms. However, the qualitative identification of a particular proteoform may not be enough to determine the biological relevance of that proteoform. Quantitative top-down MS methods have been notably applied to the study of the differing biological functions of protein proteoforms and have allowed researchers to explore proteomes at the proteoform, rather than the peptide, level. Here, we review the top-down MS methods that have been used to quantitatively identify intact proteins, discuss current applications of quantitative top-down MS analysis, and present new areas where quantitative top-down MS analysis may be implemented.

Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely character...