Identification by Shotgun Proteomics with Q-Exactive Hybrid Quadripole-Orbitrap High Resolution Tandem Mass-Spectrometry, Histone isoforms’ Hypermethylation Phenotype as A Hallmark Characteristic of Human-IDH-Mutant High-Grade Gliomas

ABSTRACT: Histone variant-post-translational modifications (PTMs) have been allied to various pathological processes, especially in cancer-onset, envisaged as obvious epigenetic biomarkers-based genotoxicity and as predictors for pathological diagnosis and prognosis. Consequently, their mapping and characterization constitute a critical field of study facilitated by the recent advances in the Mass spectrometry (MS) high-throughput technique, which has emerged as the most powerful tool to achieve insights into chromatin biology and epigenetics. The current study endeavored to the analysis of histone-isoforms’ methylation in neural cells induced by environmental stressors such as pesticides. Our protocol consisted first of a 3D in-vitro developing neurospheroid model derived from human IDH-mutant high-grade gliomas, then treatment by pesticide mixture at the inhibitory concentration IC50. Afterwards, histones were extracted, precipitated, and derivatized by propionylation prior trypsin digestion for analysis by the most popular bottom-up proteomics-based approach using a nano flow-UHPLC coupeled to Q Exactive HF hybrid quadrupole-Orbitrap high resolution tandem mass spectrometry. Raw MS files were searched against human protein database based on the species of the samples using the MAXQUANT software. The ionization was performed in a positive-ion mode and the mass spectrometer was operated in the data-dependent acquisition (DDA) mode. Histone isoforms’ methylation were validated manually by De novo peptide sequencing using the “PEAKS STUDIO” software package, which consisted in comparing the MS/MS spectra of peptide sequences between treated and non-treated spheroids. The results revealed a hypermethylation phenotype in histone isoforms, which is a hallmark characteristic of Isocitrate-Dehydrogenase (IDH)-mutated high-grade gliomas, envisaged as an adaptive strategy adapted by cancer cells to overcome stress and promote invasiveness.

INSTRUMENT(S): Ultimate 3000 nano UHPLC system (ThermoFisher Scientific, USA) with a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA), an ESI nanospray source, a higher energy collisional dissociation (HCD) fragmentation mode and data-dependent acquisition (DDA) mode for peptide sequencing.

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Kaouthar Louati  

PROVIDER: MSV000093865 | MassIVE | Sun Jan 14 09:09:00 GMT 2024

SECONDARY ACCESSION(S): PXD048530

REPOSITORIES: MassIVE