A PrSM-level Side-by-Side Comparison of On-line LCMS methods on Intact Histone H3 and H4 Proteoforms

The heterogeneity of histone H3 proteoforms has made histone H3 top-down analysis challenging. To enhance the detection coverage on the proteoforms, performing liquid chromatography (LC) front-end to MS detection is recommended. Here, by using optimized Electron-Transfer/High-Energy Collision Dissociation (EThcD) parameters, we have conducted a proteoform-spectrum match (PrSM) level side-by-side comparison on reversed-phase LC-MS (RPLC-MS), ‘Dual Gradient’ Weak Cation Exchange/Hydrophilic Interaction LC-MS (Dual Gradient WCX/HILIC-MS) and ‘Organic Rich’ WCX/HILIC-MS, on the top-down analysis of H3.1, H3.2 and H4 proteins extracted from HeLa cell culture. While both Dual Gradient WCX/HILIC and Organic Rich WCX/HILIC could resolve intact H3 and H4 proteoforms by the number of acetylations, the Organic Rich method could enhance the separations of different trimethyl/acetyl near-isobaric H3 proteoforms. In comparison with RPLC-MS, both of the WCX/HILIC-MS methods had enormously enhanced the qualities of the H3 PrSMs and remarkably improved the range, reproducibility, and confidence in the identifications of H3 proteoforms.