Monitoring the cellular metabolism of a membrane-permeant photo-caged phosphatidylinositol 3,4,5-trisphosphate derivative

Mevlut Citir, Rainer Müller, Sebastian Hauke, Alexis Traynor-Kaplan, Carsten Schultz

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

To deliver charged lipid derivatives to the cell interior, bioactivatable and photo-activatable protecting groups are frequently used. The intracellular metabolism of the protecting groups, as well as the lipid itself, are key factors that determine biological activity. Here we followed the cellular metabolism of cell-permeant photo-activatable (“caged”) and non-caged membrane-permeant analogs of dioctanoyl phosphatidylinositol 3,4,5-trisphosphate (diC8PIP3) carrying biodegradable protecting groups by mass spectrometry. After successful cell entry, the photo-activatable group can be removed on demand by a light pulse. Hence, UV irradiation acts as a switch to expose the cellular metabolism to a bolus of active compound. To investigate lipid metabolites and to capture a more complete metabolome, we adapted standard extraction methods and employed multi-reaction monitoring mass spectrometry (MRM-MS). This required a previously developed permethylation method that stabilized metabolites and enhanced volatility of the phosphoinositide metabolites. The mass spectrometric analysis allowed for the monitoring of the intracellular removal of photo-activatable caging as well as biodegradable protecting groups from the membrane-permeant phosphoinositides along with cellular turnover, namely by dephosphorylation. We found that phosphate masking groups, namely acetoxymethyl esters, were rapidly removed by endogenous enzymes while butyrates masking hydroxy groups showed a longer lifetime, giving rise to trapped intermediates. We further identified key intermediate metabolites and demonstrated the beneficial effect of caging groups and their removal on the formation of favorable metabolites. Surprisingly, caging and protecting groups were found to influence each other's stability.

Original languageEnglish (US)
Article number105124
JournalChemistry and Physics of Lipids
Volume241
DOIs
StatePublished - Nov 2021
Externally publishedYes

Keywords

  • Mass spectrometry
  • Multi-reaction monitoring
  • Phosphoinositides
  • Photoactivatable groups
  • Protecting groups

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Organic Chemistry
  • Cell Biology

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