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.

Different Modes of Action of Genetic and Chemical Downregulation of Histone Deacetylases with Respect to Plant Development and Histone Modifications

“…The procedures used for extraction and chemical derivatization of plant histones have been previously described [24]. Nuclei isolated from ~500 mg of plant tissues were resuspended in the nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 3 mM EDTA, 1% CHAPS, 0.1 μM PMSF, 45 mM NaB, and 10 μL/mL of P9599 protease inhibitor cocktail from Sigma-Aldrich), incubated for 1 h on ice, and centrifuged (8 min, 10,000× g , 4 °C).…”

“…Mass spectrometric analysis of histone peptides and following data processing was done as described previously [24]. Propionylated peptides were measured using LC-MS/MS.…”

“…These difficulties are reflected in markedly more limited utilization of MS for analyses of PTMs of plant histones compared to mammals. To overcome these obstacles, we presented a straightforward approach for preparation of plant histone samples for MS analysis based on filter-aided sample preparation coupled with histone propionylation [24].…”

Histone Acid Extraction and High Throughput Mass Spectrometry to Profile Histone Modifications in Arabidopsis thaliana

“…However, most protocols for histone extraction have been designed for non-plant organisms and produce low-purity histones or lowquality data when used in plants, evidenced by the requirement for additional purification steps, such as gel excision and chromatography, or the use of membranes to remove nonhistone proteins and other impurities in existing plant-based methods (Chen et al, 2015;L. Johnson, 2004;Ledvinová et al, 2018;Moraes et al, 2015;Zhou et al, 2021). Additional purification steps complicate the overall workflow and extend the required time to purify histones when compared to protocols used in other organisms.…”

Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype, enhances genome stability and changes chromatin compaction

“…The nuclear pellet was washed twice with 50 m m Tris‐HCl (5 min, 3000 g , 4°C). Extraction from the nuclei and chemical derivatization of plant histones were performed using previously described procedures (Ledvinova et al ., 2018). Two hundred microliters of plant histone extract in sulfuric acid was subjected to a double round of propionic anhydride derivatization, followed by clean‐up with 8 m urea (pH 8.5) on a YM‐10 Microcon filter unit (Merck Millipore, Burlington, MA, USA).…”