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A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from c...

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Autores principales: Ghosh, Avishek, Sharma, Sanjeev, Shinde, Dhananjay, Ramya, Visvanathan, Raghu, Padinjat
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822513/
https://www.ncbi.nlm.nih.gov/pubmed/31652444
http://dx.doi.org/10.1042/BSR20192502
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author Ghosh, Avishek
Sharma, Sanjeev
Shinde, Dhananjay
Ramya, Visvanathan
Raghu, Padinjat
author_facet Ghosh, Avishek
Sharma, Sanjeev
Shinde, Dhananjay
Ramya, Visvanathan
Raghu, Padinjat
author_sort Ghosh, Avishek
collection PubMed
description Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP ((18)O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P(2) using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P(2) thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.
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spelling pubmed-68225132019-11-06 A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues Ghosh, Avishek Sharma, Sanjeev Shinde, Dhananjay Ramya, Visvanathan Raghu, Padinjat Biosci Rep Cell Membranes, Excitation & Transport Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP ((18)O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P(2) using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P(2) thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo. Portland Press Ltd. 2019-10-21 /pmc/articles/PMC6822513/ /pubmed/31652444 http://dx.doi.org/10.1042/BSR20192502 Text en © 2019 The Author(s). https://creativecommons.org/licenses/by/4.0/ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).
spellingShingle Cell Membranes, Excitation & Transport
Ghosh, Avishek
Sharma, Sanjeev
Shinde, Dhananjay
Ramya, Visvanathan
Raghu, Padinjat
A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
title A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
title_full A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
title_fullStr A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
title_full_unstemmed A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
title_short A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
title_sort novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues
topic Cell Membranes, Excitation & Transport
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822513/
https://www.ncbi.nlm.nih.gov/pubmed/31652444
http://dx.doi.org/10.1042/BSR20192502
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