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Comment
. 2023 Sep 15;4(3):102226.
doi: 10.1016/j.xpro.2023.102226. Epub 2023 Aug 17.

Multiplexed targeted analysis of polyunsaturated fatty acids and oxylipins using liquid chromatography-tandem mass spectrometry

Evgenia Turtoi  1 , Jeremy Jeudy  2 , Gilles Valette  3 , Christine Enjalbal  3 , Isabelle K Vila  4 , Nadine Laguette  4 , Andrei Turtoi  5
Affiliations
Comment

Multiplexed targeted analysis of polyunsaturated fatty acids and oxylipins using liquid chromatography-tandem mass spectrometry

Evgenia Turtoi et al. STAR Protoc. .

Abstract

Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis. For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4.

Keywords: Chemistry; Mass Spectrometry; Metabolomics.

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Conflict of interest statement

Declaration of interests J.J. is employed by Agilent France, the vendor of the LC-MS system used in the present protocol.

Figures

None
Graphical abstract
Figure 1
Figure 1
Workflow of the PUFA sample preparation and analysis
Figure 2
Figure 2
SPE purification of PUFA Example of residual liquid level left above the SPE column bed to prevent sample oxidation.
Figure 3
Figure 3
Elution profiles of 12-KETE, 5-HpETE and 5-KETE from liver extracts
Figure 4
Figure 4
LC-MS chromatogram obtained with the pure standard mixture of PUFAs containing TXB2
Figure 5
Figure 5
Integration of quantifier transitions for the selected compounds The area under the curve of the peak is proportional to the concentration and is used to generate the calibration curves. Note the existence of unspecific, smaller peaks (marked with “!”).
Figure 6
Figure 6
Examples of calibration curves for selected PUFAs in the low μM range Raw data and calibration curves for 13-KODE, EPA and DHA are provided in the Figure S1.
Figure 7
Figure 7
Example of data analysis using the MetaboAnalystR package and the PUFAMetaboR script Displayed are PUFA quantification data for MDA-MB231 cancer cell-derived xenografts, following myoferlin (MYOF) gene silencing using short hairpin RNAs (shMYOF). For details see Blomme et al. (A) PLS plot outlining the control (shNT) and shMYOF tumors. (B) Volcano plot showing PUFAs with at least 2-fold changes in concentration between conditions (shNT and shMYOF). (C and D) (C) Heatmap and (D) Correlation matrix of all compounds in the dataset.
See this image and copyright information in PMC

Comment on

References

    1. Vila I.K., Chamma H., Steer A., Saccas M., Taffoni C., Turtoi E., Reinert L.S., Hussain S., Marines J., Jin L., et al. STING orchestrates the crosstalk between polyunsaturated fatty acid metabolism and inflammatory responses. Cell Metab. 2022;34:125–139.e8. - PMC - PubMed
    1. Costanza B., Turtoi A., Bellahcène A., Hirano T., Peulen O., Blomme A., Hennequière V., Mutijima E., Boniver J., Meuwis M.A., et al. Innovative methodology for the identification of soluble biomarkers in fresh tissues. Oncotarget. 2018;9:10665–10680. - PMC - PubMed
    1. Blomme A., Van Simaeys G., Doumont G., Costanza B., Bellier J., Otaka Y., Sherer F., Lovinfosse P., Boutry S., Palacios A.P., et al. Murine stroma adopts a human-like metabolic phenotype in the PDX model of colorectal cancer and liver metastases. Oncogene. 2018;37:1237–1250. - PubMed
    1. Blomme A., Costanza B., de Tullio P., Thiry M., Van Simaeys G., Boutry S., Doumont G., Di Valentin E., Hirano T., Yokobori T., et al. Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer. Oncogene. 2017;36:2116–2130. - PubMed
    1. Turtoi E., Jeudy J., Henry S., Dadi I., Valette G., Enjalbal C., Turtoi A. Analysis of Polar Primary Metabolites in Biological Samples Using Targeted Metabolomics and LC-MS. STAR Protocols. 2023 doi: 10.1016/j.xpro.2023.102400. - DOI - PMC - PubMed

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