CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - SaO-25)
High-Throughput µLC-MS/MS Lipidomics of 3D In Vitro Disease Models to Investigate Lipid Dysregulation

Darshak Gadara 1, Zdenek Spacil 1,2 *

  1. RECETOX, Masaryk University
  2. ThermoFisher Scientific, Brno

Abstrakt

Microflow liquid chromatography interfaced with mass spectrometry is increasingly applied for the high-throughput profiling of biological samples, as it demonstrated an acceptable tradeoff between sensitivity and reproducibility. However, a limited number of applications developed in lipidomics. This study introduces a robust, sensitive, and high-throughput µLC-MS/MS lipidomics workflow, applied in a differential lipidome study of APOE 3 and 4 phenotypes in iPSC-derived cerebral organoids (CO). First, we optimized the microbore column gradient conditions, injecting a mixture of 15 lipid standards on the UHPLC system coupled to a 6469 QQQ. Our µLC-MS/MS method allows quantitation of 351 lipid species from the single CO. The microflow lipidomics shows an average 3.7-fold increase in response compared to a conventional high-flow method. To benchmark the quantitative performance, 303 samples were acquired over 75 hours. The median coefficient of variation (CV) of 351 lipid species was 12.95%, only 26 lipid species show CV higher than 30%. We demonstrated that microflow separation coupled to three-stage quadruple MS substantially increases the sensitivity and allows for robust multiplexed lipid profiling (including cholesterol determination) in a single cerebral organoid. As a proof of concept, lipidomics analysis was performed for the lipid extract of APOE 3/3 and APOE 4/4 (n=10) CO samples. To our knowledge, this is the first systematic report to demonstrate the microflow LC-MS/MS allows for sensitive, high-throughput, and robust measurement of global lipidome, opening an avenue for routine application of microflow separation in the field of lipidomics.

* Korespondující autor: darshak.gadara@recetox.muni.cz


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