An experimental design to extract more information from MS-based histone studies

Clerck, Laura De; Willems, Sander; Daled, Simon; Puyvelde, Bart Van; Verhelst, Sigrid; Corveleyn, Laura; Deforce, Dieter; Dhaenens, Maarten

doi:10.1039/d1mo00201e

Cited by 3 publications

(1 citation statement)

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“…Mass spectrometry (MS)-based methods allow qualitative and quantitative characterization of protein composition of complex sample lysates. These methods also allow to identify and map posttranslational modification (PTM) of for example histone enriched protein samples (De Clerck et al, 2021;Provez et al, 2022). The modification or non-modification of an amino acid is identified by its molecular weight changing once it presents a PTM.…”

Section: Mass Spectrometry-based Methodsmentioning

confidence: 99%

Epigenetic analytical approaches in ecotoxicological aquatic research

Pham

D’Incal

et al. 2023

Environmental Pollution

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Section: Mass Spectrometry-based Methodsmentioning

confidence: 99%

Epigenetic analytical approaches in ecotoxicological aquatic research

Pham

D’Incal

et al. 2023

Environmental Pollution

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The promise of omics approaches for pediatric drug development

Verhelst,

Goessens,

Pero-Gascon

et al. 2024

Essentials of Translational Pediatric Drug Development

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Mind Your Spectra: Points to be Aware of when Validating the Identification of Isobaric Histone Peptidoforms

Hijazi,

Manessier,

Brugière

et al. 2024

Preprint

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Mass spectrometry (MS) has become a central technique to identify and quantify post-translational modifications (PTMs), overcoming limitations of antibody-based methods. Histones get dynamically modified by diverse chemical groups, particularly on their numerous lysine residues, to fine-tune all DNA-templated processes. Reliable identification of histone PTMs remains challenging and still requires manual data curation. This study focused on the Lys27-Arg40 stretch of histone H3, considered four sequence variants, an increasing number of lysine PTMs and artifacts coming from histone sample processing, which resulted in many peptides with the same atomic composition. Our analysis revealed the value of low-mass b1 and cyclic immonium fragment ions to validate identification of the distinct peptidoforms. We examined how MS/MS spectra are transformed by common software tools during the conversion of RAW files into peak lists, and highlighted how some parameters may erase the informative low-mass fragments. We established the fragmentation profiles and retention times for forty H3 K27-R40 variant x PTM combinations, including the mouse-specific variants H3mm7 and H3mm13, and targeted their detection in histone samples extracted from mouse testis and brain via a scheduled parallel reaction monitoring (PRM) analysis. The transcripts of these two mouse-specific variants were reported to be highly abundant in these tissues and the corresponding proteins may seem to be identified by data-dependent analyses. However, we only detected very low levels of the unmodified form of H3mm7 and found no trace of H3mm13 by PRM. Our work contributes to reliably deciphering the histone code shaped by distinct sequence variants and numerous combinations of PTMs.

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An experimental design to extract more information from MS-based histone studies

Abstract: Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into...

Cited by 3 publications

References 49 publications

Epigenetic analytical approaches in ecotoxicological aquatic research

Epigenetic analytical approaches in ecotoxicological aquatic research

The promise of omics approaches for pediatric drug development

Mind Your Spectra: Points to be Aware of when Validating the Identification of Isobaric Histone Peptidoforms

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