Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.

Global mapping of post-translational modifications on histone H3 variants in mouse testes.

Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.

Characterization of post-translational modifications on lysine 9 of histone H3 variants in mouse testis using matrix-assisted laser desorption/ionization in-source decay.

RATIONALE: Post-translational modifications (PTMs) of histones result in changes to transcriptional activities and chromatin remodeling. Lysine 9 of histone H3 (H3K9) is subject to PTMs, such as methylation and acetylation, which influence histone activity during spermatogenesis. Characterization strategies for studying PTMs on H3K9 have been developed to provide epigenetic and proteomic information. Proteomic analysis has been used to limited success to study PTMs on H3K9; however, a comprehensive analytical approach is required to elucidate global patterns of PTMs of H3 variants during spermatogenesis. METHODS: Intact H3 variants in mouse testis were separated by high-performance liquid chromatography on a reversed-phase column with an ion-pairing reagent. Modifications to H3K9 were identified via top-down analysis using matrix-assisted laser desorption/ionization in source decay (MALDI-ISD). RESULTS: Mono-, di-, and tri-methylations were identified at H3K9 in mouse testis and epididymis. These modifications were also observed in testis-specific histone H3 (H3t). Specifically, tri-methylation was more abundant on H3tK9 than on K9 of other H3 variants. CONCLUSIONS: We introduce a method for rapid, simple, and comprehensive characterization of PTMs on the N-termini of H3 variants using MALDI-ISD. This approach provides novel and useful information, including K9 modifications on H3t, which would benefit epigenetic and proteomic research. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

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

Various histones, including testis-specific histones, exist during spermatogenesis and some of them have been reported to play a key role in chromatin remodeling. Mass spectrometry (MS)-based characterization has become the important step to understand histone structures. Although individual histones or partial histone variant groups have been characterized, the comprehensive analysis of histone variants has not yet been conducted in the mouse testis. Here, we present the comprehensive separation and characterization of histone variants from mouse testes by a top-down approach using MS. Histone variants were successfully separated on a reversed phase column using high performance liquid chromatography (HPLC) with an ion-pairing reagent. Increasing concentrations of testis-specific histones were observed in the mouse testis and some somatic histones increased in the epididymis. Specifically, the increase of mass abundance in H3.2 in the epididymis was inversely proportional to the decrease in H3t in the testis, which was approximately 80%. The top-down characterization of intact histone variants in the mouse testis was performed using LC-MS/MS. The masses of separated histone variants and their expected post-translation modifications were calculated by performing deconvolution with information taken from the database. TH2A, TH2B and H3t were characterized by MS/MS fragmentation. Our approach provides comprehensive knowledge for identification of histone variants in the mouse testis that will contribute to the structural and functional research of histone variants during spermatogenesis.

Disruption of Th2a and Th2b genes causes defects in spermatogenesis.

The variant histones TH2A and TH2B are abundant in the testis, but their roles in spermatogenesis remain elusive. Here, we show that male mutant mice lacking both Th2a and Th2b genes were sterile, with few sperm in the epididymis. In the mutant testis, the lack of TH2B was compensated for by overexpression of H2B, whereas overexpression of H2A was not observed, indicating a decrease in the total histone level. Mutant mice exhibited two defects: incomplete release of cohesin at interkinesis after meiosis I and histone replacement during spermiogenesis. In the mutant testis, secondary spermatocytes at interkinesis accumulated and cohesin was not released normally, suggesting that the retained cohesion of sister chromatids delayed the subsequent entry into meiosis II. In addition, impaired chromatin incorporation of TNP2 and degenerated spermatids were observed in the mutant testis. These results suggest that a loss of TH2A and TH2B function in chromatin dynamics or a decrease in the total histone levels causes defects in both cohesin release and histone replacement during spermatogenesis.

Inhibitory effects of Cnidium monnieri fruit extract on pulmonary inflammation in mice induced by cigarette smoke condensate and lipopolysaccharide.

The aim of this study was to investigate the inhibitory effect of Cnidium monnieri fruit (CM) extracts on pulmonary inflammation induced in mice by cigarette smoke condensate (CSC) and lipopolysaccharide (LPS). Pulmonary inflammation was induced by intratracheal instillation of LPS and CSC five times within 12 days. CM extract was administered orally at a dose of 50 or 200 mg·kg(-1). The number of inflammatory cells in the bronchoalveolar lavage fluid was counted using a fluorescence activated cell sorter. Inflammatory mediator levels were determined by enzyme-linked immunosorbent assay. The administration of LPS and CSC exacerbated airway hyper-responsiveness (AHR) and induced an accumulation of inflammatory cells and mediators, and led to histological changes. However, these responses are modulated by treatment with CM, and the treatment with CM extract produces similar or more extensive results than the treatment with cyclosporin A (CSA). CM extract may have an inhibitory effect on pulmonary inflammation related with chronic obstructive pulmonary disease.