Development of a High-Throughput Platform for Quantitation of Histone Modifications on a New QTOF Instrument

Abstract

Histone post-translational modifications (PTMs) regulate gene expression patterns through epigenetic mechanisms. The five histone proteins (H1, H2A, H2B, H3, and H4) are extensively modified, with over 75 distinct modification types spanning more than 200 sites. Despite strong advances in mass spectrometry (MS)-based approaches, identification and quantification of modified histone peptides remains challenging because of factors, such as isobaric peptides, pseudo-isobaric PTMs, and low stoichiometry of certain marks. Here, we describe the development of a new high-throughput method to identify and quantify over 150 modified histone peptides by LC-MS. Fast gradient microflow liquid chromatography and variable window sequential windows acquisition of all theoretical spectra data-independent acquisition on a new quadrupole time-of-flight platform is compared to a previous method using nanoflow LC-MS on an Orbitrap hybrid. Histones extracted from cells treated with either a histone deacetylase inhibitor or transforming growth factor-beta 1 were analyzed by data-independent acquisition on two mass spectrometers: an Orbitrap Exploris 240 with a 55-min nanoflow LC gradient and the SCIEX ZenoTOF 7600 with a 10-min microflow gradient. To demonstrate the reproducibility and speed advantage of the method, 100 consecutive injections of one sample were performed in less than 2 days on the quadrupole time-of-flight platform. The result is the comprehensive characterization of histone PTMs achieved in less than 20 min of total run time using only 200 ng of sample. Results for drug-treated histone samples are comparable to those produced by the previous method and can be achieved using less than one-third of the instrument time.

Keywords: DIA; epigenetics; histone; mass spectrometry; post-translational modification.

Graphical abstract

Fig. 1

SWATH DIA scheme and LC gradients.A, distribution of histone peptides across the mass range. B, SWATH mass isolation scheme for histone peptides consisting of 42 variable width windows. C, DIA scheme previously utilized on the Orbitrap Exploris, consisting of 35 fixed-width windows with 1 Da overlap. D, 5-min and 10-min linear microflow gradients developed for SWATH DIA on the ZenoTOF and 55-min nanoflow gradient used for analysis on the Orbitrap Exploris. DIA, data-independent acquisition; SWATH, sequential windows acquisition of all theoretical spectra.

Fig. 2

Identification and quantification of histone peptides in H1437 cells with ZenoSWATH DIA and fast microflow gradient.A, number of peptides identified by EpiProfile 2.2 in three biological replicates analyzed with the 55-min nanoflow gradient on the Orbitrap Exploris or the 5- or 10-min microflow gradient on the ZenoTOF. B, number of peptides identified by EpiProfile 2.2 in each method with CV below 20% and below 5% for peptide retention time, peak area, and peptide ratio. C, average points across the peak for histone peptides in each of the three biological replicates by each method. Values were extracted using Skyline. D, modified histone peptides identified with the 10-min gradient ZenoTOF method, with average observed peptide ratios. E, average retention time of histone peptides observed using the 10-min microflow gradient. DIA, data-independent acquisition.

Fig 3

Accurate quantification of histone peptides in ultrafast gradients using an updated version of EpiProfile.A, top, doubly charged H4 4–17 peptide bearing a single acetylation. Bottom, fragment ions produced by H4K8ac. Labeled ions y₁₀⁺ and b₁₃⁺ are useful for distinguishing peptide isomers. Data were visualized using Skyline. B, elution profiles and quantification of histone H4 4–17 peptides bearing a single acetylation at distinct sites by EpiProfile.

Fig. 4

Differentially expressed histone PTM marks for HDACi-treated samples.A and B, volcano plots illustrating differentially expressed histone PTM marks for entinostat–control samples observed using the 10-min and 55-min methods. Changes in log2 (entinostat/control) in PTM abundance on the x-axis are plotted against the significance level represented by p value on a log10 scale on the y-axis. The dashed line denotes a fold change of 2 in either direction, whereas the horizontal line indicates the significance threshold (p < 0.05). Metaboanalyst 6.0 was used for this plot. C and D, heatmaps generated through hierarchical clustering were used to compare control and samples with two treatment conditions, M344 and entinostat, targeting HDAC inhibition observed using either the 10-min gradient ZenoTOF method or the 55-min gradient Orbitrap method. Metabolanalyst 6.0 was used for this analysis. Study samples are shown on the horizontal axis, whereas the vertical axis represents global histone PTM marks. Control is denoted by red square treated with blue and entinostat-treated samples with green. The color spectrum from dark blue to dark red represents varying expression levels from low to high. HDACi, histone deacetylase inhibitor; PTM, post-translational modification.

Fig. 5

Identification and quantification of histone peptides in HeLa cells by each method.A, the number of peptides identified in each sample (control, M344 treated, and entinostat treated) by each LC–MS method. B, correlation heatmap of all replicates of control, M344, and entinostat. C, the number of peptides identified by each method with a retention time CV below 20% and below 5%, and the number of peptides with peptide ratio CV below 20% and below 5% across all control replicates. D, correlation analysis of control, M344, and entinostat between the 10-min ZenoTOF method and 55-min Orbitrap method.

Fig. 6

Altered histone PTM marks in TGF-β-treated samples. Calculated ratios for single histone PTMs as observed using either the previous 55-min Orbitrap Exploris method or the 10-min ZenoTOF 7600 method. A and B, the ZenoTOF method recapitulates both the significant difference in expression and the peptide ratio of relatively abundant marks H3K27me3 and H3K14ac. C–E, changes in the ratios of less abundant histone PTM marks H3K27ac, H4K20me3, and H4K20me1 can be observed using the 10-min ZenoTOF method. F, changes in the level of H3K9ac, an extremely low-level mark with an isomer complicating identification and quantification, are not consistently observed across the two methods. PTM, post-translational modification; TGF-β, transforming growth factor beta.

Fig. 7

Identifications by injection amount and LC–MS method.A, number of peptides identified in each control and TGF-β treated biological replicate by injection of either 200 ng or 1 μg on the Orbitrap and 200 ng on the ZenoTOF. B, number of peptides identified in control samples with peptide ratio, area, and retention time CVs below 5% and below 20% for each method and injection amount. C, area of H3K27ac peptide observed in each control and TGF-β treated sample by method and injection amount. D, area of H3K9ac peptide observed in each sample by method and injection amount. TGF-β, transforming growth factor beta.

Fig. 8

About 100 LC–MS analyses of a histone peptide sample in <2 days.A, total number of peptides identified in each run. B, number of peptides with a CV below 20% and below 5% as calculated using observed retention times, peak areas, or peptide ratios. C, overlaid TIC for 50 consecutive injections. D, heat map chromatogram for TIC of all 100 runs. TIC, total ion chromatogram.