Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors

Abstract

Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.

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

“…The performance of histone derivatization procedure in complex plant samples was evaluated. The derivatization protocol was included into histone sample preparation workflow described in the previous study of Brabencová et al (2017) (the complete workflow is described and schematically illustrated in Figure 1 ). Unlike smooth histone preparation from mammalian cell cultures, histone extraction from plant tissues for LC-MS/MS is more challenging due to the presence of plant species-specific contaminants such as proteins, polysaccharides, polyphenols, and secondary metabolites.…”

“…In addition, it is useful for removing excess of reagent used for chemical derivatization of proteins ( Wiśniewski and Pruś, 2015 ; Vitko et al, 2016 ). In comparison to mammalian histones of relatively high purity, plant extracts are usually more complex, and their protein composition vary considerably depending on the way of sample preparation used ( Brabencová et al, 2017 ). Indeed, an inappropriate histone:reagent ratio may lead to either a high amount of residual unreacted reagent or insufficient derivatization.…”

“…The main differences between the protocols are summarized in Supplementary Table 3 . The PROP-in-SOL is preferred approach for histone preparation due to lower inter-sample variability provided which is a crucial parameter for evaluation of changes in hPTM related to certain biological basics ( Brabencová et al, 2017 ). For example, PROP-in-SOL method is more suitable for histone sample preparation from low amount of input material, i.e., young seedlings.…”

“…In the present study, we established a protocol for preparing samples of plant histones prior to LC-MS/MS analysis, using a filter-aided sample preparation (FASP) technique to remove natural contaminants and chemical additives incompatible with MS-based procedures ( Wiśniewski et al, 2009 ). We included the derivatization protocol into histone sample preparation workflow described in our previous study ( Brabencová et al, 2017 ). The final workflow consists of nuclei isolation, histone extraction into acidic solvent, protein propionylation and on-membrane digestion, then peptide propionylation in solution.…”

A Review Grinding : Teknik dan Prinsip Dasar pada Pengolahan Material

“…Salah satu tahapan yang paling penting dalam pengolahan material adalah preparasi, karena ketepatan reduksi material akan menentukan tahapan berikutnya dimana yang akan memisahkan mineral berharga dari mineral pengikitnya atau zat pengotornya (45)(46)(47). Tahap preparasi dalam mereduksi ukuran material akan dilakukan melalui proses grinding (48).…”