Evaluation of Protein Ion Relative Ratio Quantification in Top-down Electrospray Ionization-Mass Spectrometry Using...

Electrospray ionization (ESI)-mass spectrometry (MS) is a key platform for analyzing post-translationally modified proteins. With continuous advances in MS instruments and data analysis methods, top-down analysis of intact proteoforms has become highly feasible. To accurately quantify proteoforms with varying post-translational modifications (PTMs), the influence of PTMs on the ESI-MS detection efficiency must be considered. Two decades ago, Kelleher and co-workers proposed using protein ion relative ratios (PIRRs) and fragment ion relative ratios (FIRRs) in ESI-MS for proteoform quantification. While FIRR quantification has been extensively studied, the reliability of PIRR quantification─particularly for proteoforms with varying PTM degrees─remains under-evaluated. In this study, we further validated the fidelity of PIRR quantification in top-down ESI-MS using various site-specifically acetylated recombinant histone H3 proteoforms. These proteoforms, carrying varied degrees of acetylation, were produced using an orthogonal translation system that incorporates acetyllysine at the amber stop codons. After absolute quantification by UV spectrophotometry, samples were mixed in isometric ratios and analyzed by either direct infusion-ESI-MS or weak cation exchange/hydrophilic interaction-ESI-MS. Our results show that PIRRs match theoretical ratios regardless of the acetylation degree or site. These findings reinforce the validity of top-down proteoform quantification, especially for histone proteins.