ArticleBio-protocol2023
A New, Rapid Method for the Quantification of Dolichyl Phosphates in Cell Cultures Using TMSD Methylation Combined with LC-MS Analysis.
Article in Bio-protocol, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Dolichyl phosphates (DolP) are ubiquitous lipids that are present in almost all eukaryotic membranes. They play a key role in several protein glycosylation pathways and the formation of glycosylphosphatidylinositol anchors. These lipids constitute only ~0.1% of total phospholipids, and their analysis by reverse phase (RP) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is challenging due to their high lipophilicity (log P > 20), poor ionization efficiency, and relatively low abundance. To overcome these challenges, we have introduced a new approach for DolP analysis by combining trimethylsilyldiazomethane (TMSD)-based phosphate methylation and HRMS analysis. The analytical method was validated for its reproducibility, sensitivity, and accuracy. The established workflow was successfully applied for the simultaneous characterization and quantification of DolP species with different isoprene units in lipid extracts of HeLa and
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