Lipidomic Analysis of Extracellular Vesicles

The MDMS-SL assay [26 (link),58 (link),59 (link)] was performed to determine differences in lipid composition of EVs isolated from young and old WT mice using the ExoQuick protocol described above. Quantitative analysis was performed on a triple-quadruple mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an automated nanospray apparatus NanoMate and Xcalibur system. Internal standards for quantification of individual molecular species of the major lipid classes were added to each sample prior to extraction. Lipid extraction was performed via the utilization of the methyl-tert-butyl ether (MTBE) method with resuspension in a chloroform/methanol (1:1 v/v) solution with nitrogen flush. The identification and quantification of all reported lipid molecular species scans from the mass spectrometer was automatically performed with a customized sequence subroutine operated under Xcalibur software (v4.3, ThermoFisher) [58 (link),59 (link)]. The resulting MDMS-SL data were normalized to total protein content, which was assessed according to the Pierce BCA Protein Assay Kit (ThermoFisher). Abbreviations used for the classes assessed include: FA: fatty acyl chains in TAG, TAG: triacylglycerol; PC: phosphatidylcholine; PE: phosphatidylethanolamine; CAR: carnitine and acetyl carnitine; SM: sphingomyelin; CE: ceramide.

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Fitz N.F., Sahu A., Lu Y., Ambrosio F., Lefterov I, & Koldamova R. (2023). Extracellular Vesicles in Young Serum Contribute to the Restoration of Age-Related Brain Transcriptomes and Cognition in Old Mice. International Journal of Molecular Sciences, 24(16), 12550.

Publication 2023

triple-quadruple mass spectrometer NanoMate Xcalibur system Xcalibur software Pierce BCA Protein Assay Kit

Acetyl carnitine Assay Carnitine Ceramide Chloroform Flush Lipid Mdms Methanol Methyl tert butyl ether Mice Nitrogen Phosphatidylcholine Phosphatidylethanolamine Protein Scans Sphingomyelin Triacylglycerol

Corresponding Organization : University of Pittsburgh

Other organizations : Harvard University, Scripps Research Institute

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Variable analysis

independent variables

Age (young vs. old) of WT mice

dependent variables

Lipid composition of extracellular vesicles (EVs) isolated from WT mice

control variables

Total protein content (normalized to)

controls

Internal standards for quantification of individual molecular species of the major lipid classes were added to each sample prior to extraction.

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