Mass spectrometry analysis of dynamic post-translational modifications of TH2B during spermatogenesis

Abstract

TH2B, an important testis histone, plays a key role in remodeling chromatin structure during spermatogenesis. We present a detailed study of post-translational modifications (PTMs) of histone TH2B from different developmental stages of sperm cells, using a combination of high performance liquid chromatography, enzymatic Glu-c digestions of peptides, liquid chromatography-mass spectrometry (LC-MS) and LC-MS/MS analysis. The results showed modification patterns of the intact histone TH2B during spermatogenesis. Acetylated TH2B was most abundant in spermatogonia (28.9%) when compared with the spermatocytes (8.3%) and round spermatids (11.2%). Several new PTMs of TH2B were identified. In spermatogonia, spermatocytes and round spermatids, T116 and K117, were modified by phosphorylation and methylation, respectively, forming a novel 'phospho switch' site. The identified modification patterns of histone TH2B in spermatogenic cells provides a basis for future studies on histone coding and epigenetic regulation during spermatogenesis.

Disruption ofTh2aandTh2bgenes causes defects in spermatogenesis

“…4; supplementary material Table S1). This is consistent with the report that TH2B levels in spermatocytes and spermatids are approximately twofold higher than the H2B level (Lu et al, 2009). However, the H2A levels were not significantly different in the mutant cells.…”

Proteomic characterization of histone variants in the mouse testis by mass spectrometry-based top-down analysis

“…To understand their structural patterns related to chromatin dynamics, an MS-based characterization process is required. Some studies have reported the MSbased characterization on individual histone variants (4,6,7). However, it is difficult to find the comprehensive identification of intact histones in the mouse testis.…”

“…Some studies on the characterization of individual testis-specific histones during spermatogenesis have been reported. Lu et al discovered the mass value for TH2B from mice as well as those for different TH2B post-translational modifications (PTMs) present during spermatogenesis using Liquid chromatography (LC)-mass spectrometry (MS) with trypsin digestion (6). Additionally, studies have reported that LC-MS enabled characterization of TH2B expression patterns during mouse spermatogenesis (4,7).…”

Middle-down electron capture dissociation and electron transfer dissociation for histone analysis

“…Thus, even if a peptide is detected and characterized, it would be impossible to infer from which proteoform the PTMs originated from. Both AspN and GluC have been successfully used to generate middle-down peptides from histones (Kalli et al 2013;Bonenfant et al 2006;da Cunha et al 2006;Guedes et al 2011;Jung et al 2013;Kawasaki et al 2003;Lu et al 2009;Kalli and Hess 2012;Phanstiel et al 2008). The use of weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) enabled the separation of histones based on their charge (acetylation status) and hydrophilic interactions (methylation status) (Young et al 2009;Young et al 2010).…”