Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones

Abstract

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three μg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (μef) of unmodified histone proteoforms can be predicted accurately (R 2 = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

“…We concluded that CZE-MS produced comparable S/N of proteoforms to nanoRPLC-MS with 10-times lower sample consumption . More recently, we also showed that CZE-MS/MS produced comparable numbers of proteoform IDs to nanoRPLC-MS/MS for a histone sample with more than 30-fold less sample consumption (Chen et al, 2021). The studies demonstrate the better sensitivity of CZE-MS/MS for TDP compared to the widely used nanoRPLC-MS/MS, which also agrees with one previous work (Han, Wang, Aslanian, Fonslow et al, 2014).…”

“…For proteoforms with single phosphorylation and/or N‐terminal acetylation, by simply decreasing the Q by 1 charge unit for one PTM or 2 for both PTMs, the predicted and experimental µ ef of proteoforms with these PTMs had a good linear correlation ( R 2 = 0.92). The µ ef prediction model with simple modifications also showed good performance for histone proteoforms without PTMs, producing a linear correlation between predicted and experimental µ ef of those proteoforms ( R 2 = 0.98) (Chen et al, 2021). More studies about predicting µ ef of proteoforms with various PTMs, especially histone proteoforms, will be useful for understanding how PTMs influence µ ef of proteoforms and employing the predicted µ ef to evaluate the confidence of proteoform IDs.…”

“…Third, CZE‐MS has shown 10‐100 times better sensitivity than RPLC‐MS for the measurement of peptides and proteoforms (Faserl et al, 2011; Han, Wang, Aslanian, Fonslow et al, 2014; McCool & Sun, 2019; Wang et al, 2012). Lastly, the µ ef of peptides and proteoforms could be predicted accurately, potentially benefiting the identification of proteoforms with PTMs (e.g., phosphorylation and acetylation) (Chen et al, 2019, 2021; Chen, Lubeckyj et al, 2020; Kim et al, 2002; Krokhin et al, 2017).…”

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid‐2018–2022)

“…Analysis of samples containing ∼1 pg of protein digest by CE-HRMS resulted in the quantification of 157 different proteins from three different dopaminergic neurons. CZE-MS/MS was employed for top-down proteomics of calf histones [145]. Histones are important structural components of nucleoprotein chromatin that regulate many essential nuclear processes, such as DNA replication, DNA damage repair, and gene transcription.…”

Universal Automated Immunoaffinity Purification-CE−MS Platform for Accelerating Next Generation Biologic Design

“…Capillary electrophoresis (CE) has been widely used in the characterization of purified biotherapeutics for measuring critical quality attributes such as purity, charge heterogeneity, and glycosylation . Numerous CE–MS applications have been reported for proteomics, therapeutic protein intact mass analysis, and charge variant analysis, as well as biotransformation studies using a newly designed CE–MS hyphenation technology. − To study biotransformation, effective sample preparation to enrich the target analytes from biological matrices is critical. Traditionally, immunoaffinity purification (immunoaffinity capture, IA) has been performed using magnetic beads labeled with a capture reagent.…”