Dataset Information

Sample preparation procedure for mass spectrometric analysis of plant histones

ABSTRACT: The protocol for preparing samples of plant histones prior to LC-MS/MS analysis was established, using a filter-aided sample preparation (FASP) technique to remove natural contaminants and chemical additives incompatible with MS-based procedures. The protocol consists of nuclei isolation, histone extraction into acidic solvent, protein propionylation and on-membrane digestion, then peptide propionylation in solution. Two variants of the derivatization protocol, in-solution propionylation in the vial (PROP-in-SOL) and propionylation on the filter unit (PROP-on-FILTER), were compared by efficiency of the labeling and by abundance of identified histone peptide forms.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell

SUBMITTER: Gabriela Lochmanova

LAB HEAD: Zbynek Zdrahal

PROVIDER: PXD010102 | Pride | 2018-09-20

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Ath_Filter_Orbi-1_prop120min-IS_wt_1_Recal.pep.xml	Pepxml
	Ath_Filter_Orbi-1_prop120min-IS_wt_1_Recal.prot.xml	Xml
	Ath_Filter_Orbi-1_prop120min-IS_wt_1_Recal.raw	Raw
	Ath_Filter_Orbi-1_prop120min-IS_wt_2_Recal.pep.xml	Pepxml
	Ath_Filter_Orbi-1_prop120min-IS_wt_2_Recal.prot.xml	Xml

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Publications

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

Ledvinová Dominika D Mikulášek Kamil K Kuchaříková Hana H Brabencová Sylva S Fojtová Miloslava M Zdráhal Zbyněk Z Lochmanová Gabriela G

Frontiers in plant science 20180919

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 ...[more]

PMID: 30283482

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Project description:Propionate is a common three-carbon intermediate produced from the breakdown of propiogenic substrates, such as branched-chain amino acids and odd-numbered fatty acids, and by gut bacteria. Propionate can chemically modify proteins, and if this involves histones, it may underpin a disease-relevant link between short-chain acyls and gene expression in the heart. Here, we sought to characterize how propionate-dependent modifications to histones affect cardiac gene expression and contractile function in a mouse model producing elevated levels of propionate/propionyl-CoA. An adult mouse model of propionic acidaemia (PA) was used to investigate how propionate affects histones and gene expression in the heart, an organ strongly affected in PA patients. Mass spectrometry confirmed elevated plasma propionate in 8-week PA mice, reaching levels detected in PA patient serum. Metabolomic analyses confirmed a metabolic signature of PA, but male mice had enhanced propionate processing towards -alanine. Female PA hearts had expanded end-diastolic and end-systolic volumes, and weaker systolic contractions, without major electrocardiographic changes. Ca2+ signals were deranged in female PA mice (raised diastolic Ca2+), consistent with contractile dysfunction. Differentially-expressed genes (DEGs) included Pde9a and Mme, previously linked to cardiac dysfunction. These DEGs also responded to 48-h culture of wild-type myocytes with propionate as well as butyrate, an HDAC inhibitor, suggesting a role for increased histone acetylation alongside propionylation in inducing these genes. Indeed, histone acetylation (H3K27ac) and propionylation were elevated genome-wide in the PA heart and at the promoters of Pde9a and Mme. These propionate-associated epigenetic responses were more pronounced in female PA mice. The greater prominence of epigenetic, transcriptional, and functional responses in female PA mice, despite a mostly sex-indiscriminate overall metabolic milieu, argues that histone acylation plays a defining role in the cardiac phenotype. We conclude that perturbed propionate metabolism in vivo alters histone acylation and gene expression, which impacts cardiac contractile function.