Dataset Information

Comparison of in-gel digestion methods for histone PTM analysis

ABSTRACT: In order to optimize the histone digestion procedure for low-abundance clinical samples, we compared different in-gel digestion protocols. Histones are usually digested in-gel using “Arg-C-like” strategies, which involve chemical acylation of lysines followed by trypsin digestion (17). Acylated lysines are not recognized by trypsin, which -as a consequence- cuts only at the C-terminus of arginines and generates peptides of suitable length for MS analysis. We compared four Arg-C-like protocols: 1) D3 protocol (deuterated acetic anhydride as acylating agent); 2) PRO protocol (propionic anhydride as acylating agent); 3) PRO-PRO protocol (propionic anhydride as acylating agent and peptide N-terminal derivatization); 4) PRO-PIC protocol (propionic anhydride as acylating agent and phenyl-isocyanate as peptide N-terminal derivatization). We also used an Arg-C in solution digestion as a reference, and tested the performance of the methods with decreasing sample amounts.

INSTRUMENT(S): Q Exactive Plus, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Breast

DISEASE(S): Brain Cancer,Breast Cancer

SUBMITTER: Roberta Noberini

LAB HEAD: Tiziana Bonaldi

PROVIDER: PXD024745 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples.

Noberini Roberta R Savoia Evelyn Oliva EO Brandini Stefania S Greco Francesco F Marra Francesca F Bertalot Giovanni G Pruneri Giancarlo G McDonnell Liam A LA Bonaldi Tiziana T

Clinical epigenetics 20210728 1

<h4>Background</h4>Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for subs ...[more]

PMID: 34315505

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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.

			Action	DRS
	D3_436.zip	Other
	D3_GLIO.zip	Other
	GLIO_PRO2.zip	Other
	HF180504_RN_glio_D3_1.raw	Raw
	HF180504_RN_glio_D3_2.raw	Raw