Journal
JOURNAL OF PROTEOME RESEARCH
Volume 17, Issue 11, Pages 3791-3800Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.8b00458
Keywords
histones; top-down mass spectrometry; online comprehensive two-dimensional liquid chromatography; ultraviolet photodissociation; post-translational modifications
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Funding
- Netherlands Organization for Scientific Research by the NWO Veni grant IPA [722.015.009]
- Office of Biological and Environmental Research, U.S. Department of Energy (DOE)
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Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLCXLC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLCXLC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (similar to 1.5 mu g).
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