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Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS.

ABSTRACT: We describe a protocol for measuring phospholipid class and fatty acid composition in the budding yeast Saccharomyces cerevisiae using a liquid chromatography-mass spectrometry (LC-MS)-based approach. We compile a mass spectral-retention time library verified for major phospholipids in the budding yeast and showcase the profiling process of phospholipid compositions in mutants with defective syntheses of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). We further provide methods for extracting and quantifying phospholipids in mammalian systems. For complete details on the use and execution of this protocol, please refer to Fang et al. (2022).

SUBMITTER: Yang S

PROVIDER: S-EPMC9579711 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS.

Yang Sen S Xue Jingyuan J Ye Cunqi C

STAR protocols 20221014 4

We describe a protocol for measuring phospholipid class and fatty acid composition in the budding yeast Saccharomyces cerevisiae using a liquid chromatography-mass spectrometry (LC-MS)-based approach. We compile a mass spectral-retention time library verified for major phospholipids in the budding yeast and showcase the profiling process of phospholipid compositions in mutants with defective syntheses of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). We further provide methods for e ...[more]

PMID: 36240059

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Project description:Assessing human exposure to commonly used, highly toxic, but non-persistent organophosphates (OPs) is challenging because these toxicants are readily biotransformed into dialkyl phosphates (DAPs) and other metabolites. Growing hair accumulates toxicants and their metabolites, which makes hair a valuable non-invasively sampled matrix that can be used to retrospectively examine chemical exposure. However, the efficient quantification of hydrophilic DAP compounds in hair is challenging due to complex hair matrix effects. To improve upon existing methods, we first examined the acid dissociation constants (pKa) of DAPs and amino acids (major components in hair) and identified the best pH conditions for minimizing matrix effects. We hypothesized that under basic pH conditions DAPs and amino acids would be negatively charged and have weak interactions favorable to DAP dissociation from the matrix. To test this, we compared the efficiency of various pH conditions of suitable solvents to extract six DAPs from hair samples, and we quantified these DAPs using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). As expected, a basic extraction (methanol with 2% NH4OH) approach had the highest extraction efficiency and yielded satisfactory recoveries for all six DAPs (72%-152%) without matrix effects. Additionally, the alkaline extract can be directly injected into the LC-MS/MS. This relatively rapid and simple procedure allowed us to process up to 90 samples per week with reproducible results. To our knowledge, this is the first method to quantify all six DAPs simultaneously in hair using LC-MS/MS with electrospray ionization (ESI) in negative ion mode. Finally, we demonstrated the feasibility of measuring DAP levels in hair samples from patients affected with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease potentially linked to OP exposure. Due to our optimized solvent extraction process, the method we have developed is compatible with the rapidity and sensitivity needed for hair analysis applied to population biomonitoring.

Dataset Information

Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS.

Publications

Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS.

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