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

Kwak, Ho Geun; Dohmae, Naoshi

doi:10.5582/bst.2016.01090

Cited by 14 publications

(11 citation statements)

References 23 publications

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“…For each of these protein species, the detailed information including spectrum index, retention time in minute, the precursor ion (isolation m/z , experimental m/z , theoretical m/z , z , IPMD in ppm, theoretical monoisotopic mass in Da), protein name, PTMs, number of matched product ions, P score, PTM score, and protein species score was provided in Table S1. It is worth mentioning that only 16 protein species were identified in the literature focusing on top‐down proteomics of histones from mouse …”

Section: Resultsmentioning

confidence: 99%

“…With the development of high‐resolution mass spectrometry, top‐down proteomics, which characterizes proteins at the intact level and has the strength of 100% sequence coverage, has been the method of choice for full characterization of combinatorial PTMs in core histones from various species . For mouse specifically, in 2010, the core histones from murine embryonic stem cells were separated using RPLC, analyzed by Orbitrap MS with ESI and ETD, searched by a combination of Xtract, Protein Prospector, and Fragment Assignment by Visual Assistance (FAVA); seven protein species were identified .…”

Section: Introductionmentioning

confidence: 99%

“…In 2016, mouse core histones extracted from testes and epididymides were separated using a C4 column; separated histone fractions were collected and analyzed by MS/MS with HCD on a Q‐Exactive MS. The masses of 46 histone variants and their PTMs were calculated by deconvolution according to the MS spectra; elevated ratios of testis‐specific histones were observed in the testis, and some somatic histones increased in the epididymis . In 2017, mouse brain core histones were separated on a C18 column, and tandem mass spectra using both ETD and HCD were acquired on an Orbitrap Fusion Lumos MS; with data analysis using the multiple software package (ProMex, MSPathFinder, LcMsSpectator, and TopPIC), 178 protein species with E‐value lower than 1 × 10 −40 were identified, and novel histone PTMs including tyrosine bromination on H4 and H2A as well as glutathionylation on H3 were identified …”

Section: Introductionmentioning

confidence: 99%

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Top‐down characterization of mouse core histones

2019

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Histone post-translational modifications (PTMs) play various roles in chromatinrelated cellular processes, and comprehensive analysis of these combinatorial PTMs at the intact protein level by top-down proteomics is the method of choice to reveal their crosstalk and biological functions. Here, we report our top-down characterization of the core histones from mouse fibroblasts cells NIH/3T3, which is a classic model used in many kinds of research. With nanoRPLC-MS/MS analysis and ProteinGoggle database search, 547 protein species were identified with spectrumlevel FDR ≤ 1%, where PTMs in 51 protein species were unambiguously localized with PTM scores ≥1. High-resolution MS/MS data also allowed the unambiguous identification of acetylation instead of trimethylation. This study presents a general picture of combinatorial PTMs of mouse core histones, which serves as a basic reference for all future related biological studies.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Top‐down characterization of mouse core histones

2019

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“…1 ). In our past study, we had employed MS-based analysis for verification of each separated histone H3 variant [16] , [18] . Epididymis is a tube connected to the testis where sperms flow and get temporarily stored [5] .…”

Section: Resultsmentioning

confidence: 99%

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

Kwak

Suzuki

Dohmae

2017

Biochemistry and Biophysics Reports

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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.

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“…Since the protein expression of Bub1 is maintained in MII oocytes40, it is possible that it persists in zygotes and phosphorylates TH2A T120 in the first mitosis. In addition, recent proteomic analyses of mouse testis found that the acetylation of TH2A at the N-terminus is also expected41.…”

Section: Discussionmentioning

confidence: 99%

Identification of a variant-specific phosphorylation of TH2A during spermiogenesis

Hada

Masuda

Yamaguchi

et al. 2017

Sci Rep

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Tissue-specific histone variant incorporation into chromatin plays dynamic and important roles in tissue development. Testis is one such tissue, and a number of testis-specific histone variants are expressed that have unique roles. While it is expected that such variants acquire post-transcriptional modifications to be functional, identification of variant-specific histone modifications is challenging because of the high similarity of amino acid sequences between canonical and variant versions. Here we identified a novel phosphorylation on TH2A, a germ cell-specific histone H2A variant. TH2A-Thr127 is unique to the variant and phosphorylated concomitant with chromatin condensation including spermiogenesis and early embryonic mitosis. In sperm chromatin, phosphorylated TH2A-Thr127 (=pTH2A) is co-localized with H3.3 at transcriptional starting sites of the genome, and subsequently becomes absent from the paternal genome upon fertilization. Notably, pTH2A is recurrent and accumulated in the pericentromeric heterochromatin of both paternal and maternal chromosomes in the first mitosis of embryos, suggesting its unique regulation during spermiogenesis and early embryogenesis.

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

Cited by 14 publications

References 23 publications

Top‐down characterization of mouse core histones

Top‐down characterization of mouse core histones

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

Identification of a variant-specific phosphorylation of TH2A during spermiogenesis

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