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Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography–High Resolution Mass Spectrometry
[Image: see text] Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by vario...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933046/ https://www.ncbi.nlm.nih.gov/pubmed/36716239 http://dx.doi.org/10.1021/acs.analchem.2c03623 |
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author | Kale, Dipali Kikul, Frauke Phapale, Prasad Beedgen, Lars Thiel, Christian Brügger, Britta |
author_facet | Kale, Dipali Kikul, Frauke Phapale, Prasad Beedgen, Lars Thiel, Christian Brügger, Britta |
author_sort | Kale, Dipali |
collection | PubMed |
description | [Image: see text] Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC–MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer. |
format | Online Article Text |
id | pubmed-9933046 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-99330462023-02-17 Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography–High Resolution Mass Spectrometry Kale, Dipali Kikul, Frauke Phapale, Prasad Beedgen, Lars Thiel, Christian Brügger, Britta Anal Chem [Image: see text] Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC–MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer. American Chemical Society 2023-01-30 /pmc/articles/PMC9933046/ /pubmed/36716239 http://dx.doi.org/10.1021/acs.analchem.2c03623 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Kale, Dipali Kikul, Frauke Phapale, Prasad Beedgen, Lars Thiel, Christian Brügger, Britta Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography–High Resolution Mass Spectrometry |
title | Quantification
of Dolichyl Phosphates Using Phosphate
Methylation and Reverse-Phase Liquid Chromatography–High Resolution
Mass Spectrometry |
title_full | Quantification
of Dolichyl Phosphates Using Phosphate
Methylation and Reverse-Phase Liquid Chromatography–High Resolution
Mass Spectrometry |
title_fullStr | Quantification
of Dolichyl Phosphates Using Phosphate
Methylation and Reverse-Phase Liquid Chromatography–High Resolution
Mass Spectrometry |
title_full_unstemmed | Quantification
of Dolichyl Phosphates Using Phosphate
Methylation and Reverse-Phase Liquid Chromatography–High Resolution
Mass Spectrometry |
title_short | Quantification
of Dolichyl Phosphates Using Phosphate
Methylation and Reverse-Phase Liquid Chromatography–High Resolution
Mass Spectrometry |
title_sort | quantification
of dolichyl phosphates using phosphate
methylation and reverse-phase liquid chromatography–high resolution
mass spectrometry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933046/ https://www.ncbi.nlm.nih.gov/pubmed/36716239 http://dx.doi.org/10.1021/acs.analchem.2c03623 |
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