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Analysis of phosphate and phosphate containing headgroups enzymatically cleaved from phospholipids of Bacillus subtilis by capillary electrophoresis

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Abstract

A new, fast, selective, and reliable capillary electrophoresis method has been developed for analysis of selected phosphoesters (phosphoserine, phosphoethanolamine, phosphoglycerol) and phosphate. The method is based on separation of specific phosphate containing headgroups (phosphoesters) which are cleaved from the glycerol skeleton of a phospholipid by a regioselective enzyme (phospholipase C). Analysis of intact phospholipids with the same polar headgroup but different fatty acids shows that fatty acid composition has a high impact on separation of phospholipids, so analysis of separated polar headgroups, which avoids this influence, represents a much more suitable approach for phospholipid class research. Optimization of method parameters results in running buffers of relatively narrow pH interval (pH about 10) where all phosphoesters are separated. Further method validation has shown that direct UV detection has a sufficient detection limit for all analytes to perform suitable analyses of cell membrane lipids. The optimized method was tested on the lysate of cell membrane of Bacillus subtilis, where all analytes were determined.

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Acknowledgments

This work was supported by SVV260205 and 13-18051P from the Czech Science Foundation and authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43, Prague 2, Czech Republic

    Václav Matěj Bierhanzl, Lukáš Taraba & Radomír Čabala

  2. Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43, Prague 2, Czech Republic

    Martina Riesová

  3. Institute of Forensic Medicine and Toxicology, General University Hospital in Prague, U Nemocnice 2, 128 08, Prague 2, Czech Republic

    Radomír Čabala

  4. Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44, Prague 2, Czech Republic

    Gabriela Seydlová

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  1. Václav Matěj Bierhanzl
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  2. Martina Riesová
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  3. Lukáš Taraba
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  4. Radomír Čabala
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Correspondence to Václav Matěj Bierhanzl.

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Bierhanzl, V.M., Riesová, M., Taraba, L. et al. Analysis of phosphate and phosphate containing headgroups enzymatically cleaved from phospholipids of Bacillus subtilis by capillary electrophoresis. Anal Bioanal Chem 407, 7215–7220 (2015). https://doi.org/10.1007/s00216-015-8885-x

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  • DOI: https://doi.org/10.1007/s00216-015-8885-x

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