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Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis
Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enab...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032999/ https://www.ncbi.nlm.nih.gov/pubmed/26010583 http://dx.doi.org/10.1002/pmic.201400569 |
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author | Meert, Paulien Govaert, Elisabeth Scheerlinck, Ellen Dhaenens, Maarten Deforce, Dieter |
author_facet | Meert, Paulien Govaert, Elisabeth Scheerlinck, Ellen Dhaenens, Maarten Deforce, Dieter |
author_sort | Meert, Paulien |
collection | PubMed |
description | Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom‐up MS on histones. |
format | Online Article Text |
id | pubmed-5032999 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-50329992016-10-03 Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis Meert, Paulien Govaert, Elisabeth Scheerlinck, Ellen Dhaenens, Maarten Deforce, Dieter Proteomics Cell Biology Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom‐up MS on histones. John Wiley and Sons Inc. 2015-06-22 2015-09 /pmc/articles/PMC5032999/ /pubmed/26010583 http://dx.doi.org/10.1002/pmic.201400569 Text en © 2015 The Authors. PROTEOMICS Published by Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. |
spellingShingle | Cell Biology Meert, Paulien Govaert, Elisabeth Scheerlinck, Ellen Dhaenens, Maarten Deforce, Dieter Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
title | Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
title_full | Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
title_fullStr | Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
title_full_unstemmed | Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
title_short | Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
title_sort | pitfalls in histone propionylation during bottom‐up mass spectrometry analysis |
topic | Cell Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032999/ https://www.ncbi.nlm.nih.gov/pubmed/26010583 http://dx.doi.org/10.1002/pmic.201400569 |
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