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

Abstract

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.

Chromatographic separation of peptides and proteins for characterization of proteomes

“…In addition to RPLC, several other separation modes are also combined with MS for top-down proteomic researches, including size-exclusion chromatography (SEC), [178][179][180][181] ion-exchange chromatography (IEX), [182][183][184] hydrophobic interaction chromatography (HIC), [185][186][187][188] and HILIC. [189][190][191][192][193][194] Online SEC-MS is wellsuited for analysis of native proteins or protein complexes using nondenaturing mobile phases, [178][179][180][181] but it suffers from very low-resolution separation. Online IEX-MS using a volatile salt or pH gradient has proven successful for biopharmaceutical applications.…”

“…HILIC has good compatibility with MS and good orthogonality with RPLC. Online HILIC-MS has been successfully applied for analysis of histone proteins, 190 biopharmaceuticals, 191 serum immunoglobulins 192 and glycoproteins. 193,194 For these proteins, HILIC resolved proteoforms that would coelute in RPLC.…”

Recent stationary phase‐based fractionation strategies in proteomic analysis

“…Between the two WCX‐HILIC modes, the “organic‐rich” method could separate better the near‐isobaric proteoforms (Figure 4). Overall, compared to RPCL, the WCX‐HILIX methods improved the range, reproducibility, and confidence in the identifications of H3 proteoforms [62]. The last two mentioned studies [61, 62] show the potential of WCX columns eluted with ACN‐rich and salt‐free mobile phases for the prefractionation of intact or partially digested proteins (top‐down and middle‐down studies) that could be retained both by charge and hydrophilicity.…”

“…The color of the proteoforms‐spectrum match represents their respective number of acetylations, as indicated by the color bar. Reprinted with permission, [62].…”