Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones

Abstract

Characterization of histone post-translational modifications (PTMs) is still challenging, and robust histone sample preparation is essential for convincing evaluation of PTMs by mass spectrometry. An effective protocol for extracting plant histone proteins must also avoid excessive co-extraction of the numerous potential interfering compounds, including those related to secondary metabolism. Currently, the co-existence of histone marks is addressed mostly by shotgun proteomic analysis following chemical derivatization of histone lysine residues. Here, we report a straightforward approach for plant histone sample preparation for mass spectrometry, based on filter-aided sample preparation coupled with histone propionylation. The approach offers savings in sample handling and preparation time, enables removal of interfering compounds from the sample, and does not require either precipitation or dialysis of histone extract. We show the comparison of two protocol variants for derivatization of histone proteins, in-solution propionylation in the vial and propionylation on the filter unit. For both protocols, we obtained identical abundances of post-translationally modified histone peptides. Although shorter time is required for histone protein labeling on the filter unit, in-solution derivatization slightly outweighed filter-based variant by lower data variability. Nevertheless, both protocol variants appear to be efficient and convenient approach for preparation of plant histones for mass spectrometric analysis.

Keywords: Arabidopsis thaliana; epigenetics; filter-aided sample preparation; histone derivatization; mass spectrometry; post-translational modifications.

FIGURE 1

Plant histone sample preparation workflow for LC-MS/MS. (A) An illustrative scheme of the workflow. (B) Protocol variants for protein derivatization; time needed for each derivatization protocol is depicted.

FIGURE 2

Comparison of PROP-in-SOL and PROP-on-FILTER performance. (A) Grayscale pie charts showing proportions of identified histone H3 and H4 peptides in four categories – desired (peptides cleaved and propionylated as expected), underpropionylated (peptides with at least one unmodified amino group on lysine residue or N-terminus), overpropionylated (peptides with at least one propionylated hydroxyl group on S, T, or Y residue), non-specifically cleaved (peptides with cleavage at lysine C terminus or missed cleavage at arginine C terminus). Color pie charts showing proportions of assignable peptides, i.e., peptides enabling correct quantification. (B) Box-plots and scatter-plots of the means and standard deviations of abundances of histone H3 and H4 peptide forms detected in the samples. The box-plots show extremes, interquartile ranges and medians (N = 23). Means and standard deviations were compared by Mann-Whitney tests (p-values) and Spearman’s correlation coefficients (SCC values). (C) Radar charts showing relative abundances of individual peptide forms of histone H3 and H4 (medians; N = 5), determined from the ratio of the XIC peak areas of particular assignable products to the summed XIC peak areas of the total pool of all quantified H3 or H4 peptides, respectively. The Y axes have a binary logarithm scale, with zero located in the center.

FIGURE 3

PROP-in-SOL performance for hPTM characterization in different plant developmental stages. (A) Box-plots of the means and standard deviations of abundances of histone H3 and H4 peptide forms detected in the seedling and leaf samples. The box-plots show extremes, interquartile ranges and medians (N = 21). Means and standard deviations were compared by Mann-Whitney tests (p-values). (B) Radar charts showing relative abundances of individual peptide forms of histone H3 and H4 (medians; N = 5), determined from the ratio of the XIC peak areas of particular assignable products to the summed XIC peak areas of the total pool of all quantified H3 or H4 peptides, respectively. The Y axes have a binary logarithm scale, with zero located in the center.