Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions
Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly mod...
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doaj-6df7284da78e430983bdcf2e793f5b282021-04-21T23:03:09ZengMDPI AGProteomes2227-73822021-04-019171710.3390/proteomes9020017Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed DecisionsSimon Daled0Sander Willems1Bart Van Puyvelde2Laura Corveleyn3Sigrid Verhelst4Laura De Clerck5Dieter Deforce6Maarten Dhaenens7Laboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumDepartment of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, GermanyLaboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Pharmaceutical Biotechnology/ProGenTomics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumHistone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX<sup>®</sup>, Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.https://www.mdpi.com/2227-7382/9/2/17histone codecoverageepigeneticsmass spectrometrysample preparationworkflow optimization |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Simon Daled Sander Willems Bart Van Puyvelde Laura Corveleyn Sigrid Verhelst Laura De Clerck Dieter Deforce Maarten Dhaenens |
spellingShingle |
Simon Daled Sander Willems Bart Van Puyvelde Laura Corveleyn Sigrid Verhelst Laura De Clerck Dieter Deforce Maarten Dhaenens Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions Proteomes histone code coverage epigenetics mass spectrometry sample preparation workflow optimization |
author_facet |
Simon Daled Sander Willems Bart Van Puyvelde Laura Corveleyn Sigrid Verhelst Laura De Clerck Dieter Deforce Maarten Dhaenens |
author_sort |
Simon Daled |
title |
Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions |
title_short |
Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions |
title_full |
Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions |
title_fullStr |
Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions |
title_full_unstemmed |
Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions |
title_sort |
histone sample preparation for bottom-up mass spectrometry: a roadmap to informed decisions |
publisher |
MDPI AG |
series |
Proteomes |
issn |
2227-7382 |
publishDate |
2021-04-01 |
description |
Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX<sup>®</sup>, Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423. |
topic |
histone code coverage epigenetics mass spectrometry sample preparation workflow optimization |
url |
https://www.mdpi.com/2227-7382/9/2/17 |
work_keys_str_mv |
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