PrSM-Level Side-by-Side Comparison of Online LC-MS Methods with Intact Histone H3 and H4 Proteoforms

The
heterogeneity of histone H3 proteoforms makes histone H3 top-down
analysis challenging. To enhance the detection coverage of the proteoforms,
performing liquid chromatography (LC) front-end to mass spectrometry
(MS) detection is recommended. Here, using optimized electron-transfer/high-energy
collision dissociation (EThcD) parameters, we have conducted a proteoform-spectrum
match (PrSM)-level side-by-side comparison of 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 analyses of H3.1, H3.2, and H4 proteins
extracted from a 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 enhanced
the qualities of the H3 PrSMs and remarkably improved the range, reproducibility,
and confidence in the identifications of H3 proteoforms.