A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling

Abstract

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d 0 -acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.

Collision energies: Optimization strategies for bottom‐up proteomics

“…MS/MS measurements of synthetic dimethylarginine peptides and endogenous methyl-peptides enriched from HEK 293 T cell lysate on Thermo Orbitrap Fusion Lumos and Q Exactive Plus mass spectrometers showed optimal identification and annotation of dimethylarginine peptides at 32 NCE% (Fusion Lumos) and 35 NCE% (Q Exactive) (Hartel et al, 2020). The increased neutral loss from dimethylarginine peptides at elevated CE (38 NCE% on a Q Exactive) was corroborated by Zappacosta et al (2021).…”

Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry

“…Acetylation also improved the detection sensitivity of sterols in thermospray ionization [53]. The positive effect of acetylation was also observed with electrospray ionization [54][55][56][57]. Acetylated peptides showed improved fragmentation patterns, allowing for better sequence information [56].…”

“…The intensities of b [54,55] and y ions increased [56], while less informative fragments were suppressed. Acetylated peptides were used for de novo sequencing [55,57]. Acetylation is also used to improve analyte detection in APPI-MS [58].…”

“…Acetylated peptides showed improved fragmentation patterns, allowing for better sequence information [56]. The intensities of b [54,55] and y ions increased [56], while less informative fragments were suppressed. Acetylated peptides were used for de novo sequencing [55,57].…”