Development of a high-throughput (10 minute microflow gradient) platform for quantitation of histone modifications on a new QTOF instrument

ABSTRACT: Histone post-translational modifications (PTMs) regulate gene expression patterns through epigenetic mechanisms. The 5 histone proteins (H1, H2A, H2B, H3, and H4) are extensively modified, with over 75 distinct modification types spanning more than 200 sites. Despite strong advances in mass spectrometry based approaches, identification and quantification of modified histone peptides remains challenging due to factors such as isobaric peptides, pseudo-isobaric PTMs, and low stoichiometry of certain marks. Here we describe the development of a new high-throughput method to identify and quantitate over 150 modified histone peptides by liquid chromatography-mass spectrometry (LC-MS). Fast gradient microflow liquid chromatography and variable window SWATH data-independent acquisition on a new quadrupole time-of-flight platform is compared to a previous method using nanoflow LC-MS on an Orbitrap hybrid instrument. Histones extracted from cells treated with either a histone deacetylase inhibitor (HDACi) or TGF-beta 1 were analyzed by data-independent acquisition (DIA) on two mass spectrometers: an Orbitrap Exploris 240 with a 55 minute nanoflow LC gradient, and the SCIEX ZenoTOF 7600 with 5 and 10 minute microflow gradients, with data analyzed using newly developed in-house software. To demonstrate the reproducibility and speed advantage of the method, 100 consecutive injections of one sample were performed in less than 2 days on the QTOF platform. The result is the comprehensive characterization of histone PTMs achieved in less than 20 minutes of total run time using only 200 ng of sample. Results for histone PTMs extracted from cells treated with epigenetic drugs or undergoing cellular transformation are comparable to those produced by the previous method, but can be achieved using less than one-third of the instrument time and one-fifth of the sample amount.

ORGANISM(S): Homo Sapiens

SUBMITTER: Francisca N de Luna Vitorino  

PROVIDER: PXD056434 | panorama | Fri Oct 31 00:00:00 GMT 2025

REPOSITORIES: PanoramaPublic