Evaluation of Protein Ion Relative Ratio Quantification in Top-down Electrospray Ionization–Mass Spectrometry Using Site-Specific Acetylated Recombinant Histone H3 Proteoforms

ABSTRACT: Electrospray ionization (ESI)-mass spectrometry (MS) provides a crucial platform for analyzing post-translationally modified proteins. With continuous advances in MS instruments and data analysis approaches, analyzing intact proteoforms using a “top-down” approach has become highly feasible. To quantify proteoforms varied in post-translational modification (PTM), the PTMs’ influence on ESI-MS detection efficiency must be addressed. Two decades ago, the con-cept of using protein ion relative ratios (PIRRs) and fragment ion relative ratios (FIRRs) in ESI-MS for proteoform quantification was proposed by Kelleher and co-workers. Over these two decades, while FIRR quantification has been vigorously studied, the reliability of PIRR quantifi-cation, particularly for proteoforms varied in PTM extents, is relatively under-evaluated. In this study, using various site-specifically acetylated recombinant histone H3 proteoforms, the fidelity of PIRR quantification in top-down ESI-MS is further documented. Using an acetyllysine-amber stop codon orthogonal translation system, recombinant histone H3 carrying various number of acetylation were produced. They were subjected to absolute quantification using UV spectropho-tometry and combined in isometric ratio before analysis by either direct infusion-ESI-MS or weak cation exchange/hydrophilic interaction-ESI-MS. We report that all of the measured PIRRs closely reflect their theoretical ratios, regardless of acetylation extents and locations. These re-sults well supplement the supports on the validity of top-down proteoform quantification, partic-ularly for histone proteins.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kin Wing Lui  

LAB HEAD: Ting Fung Chan

PROVIDER: PXD064777 | Pride | 2025-09-29

REPOSITORIES: Pride