Ingvild Wathne,a, Kine Ø. Hansen a, Johan M. Isaksson,a,b and Terje Vasskog,a
a Natural Products and Medicinal Chemistry Research Group, Department of Pharmacy, UiT – the Arctic University
of Norway
b The Chemical Synthesis and Analysis, Department of Chemistry, UiT – the Arctic University of Norway
Email: Ingvild.wathne@uit.no
Lipidomics is the comprehensive study of the lipid content within a sample, and lipid research is crucial in biological and biomedical sciences by improving our understanding of cellular processes, signalling pathways, and disease mechanisms (1). Lipidomics can be performed by various analytical tools, but high-performance liquid chromatography – mass spectrometry (HPLC-MS) is often preferred as it enables sensitive, high-throughput analysis of intact lipid species and allows for comparison of relative abundance across samples. However, a key limitation to this approach is that signal intensities cannot be directly compared across different lipid classes due to variation in ionisation efficiency. This means that while it is possible to compare relative lipid levels between samples, accurately comparing the abundance of different lipid classes within the same sample is unreliable without the use of internal standards which would significantly add to the cost and time consumption of the analysis. Although pure lipid standards can be used to measure ionisation efficiency under controlled conditions, such measurements do not account for matrix effects such as ion suppression, that occur in complex biological samples (2). As a complementary strategy, phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy can be used to quantify phospholipid head groups, revealing the true ratio between different phospholipids (3).
The aim of this project was to establish a correlation between the relative quantification of lipid classes in complex samples by UHPLC-MS and 31P NMR. For initial development of the method, herring roe was used as the sample material for lipid extraction. Lipids were extracted in triplicate using a modified solid-liquid extraction protocol developed by Folch et al. (4). The lipid extracts were analysed by UHPLC-HRMSn with AcquireX-based data-dependent acquisition. Data was processed in LipidSearch, enabling identification and relative quantification of lipid classes and individual lipid species. The same extracts were subsequently analysed by 31P NMR spectroscopy. Phospholipid classes were identified by comparing the chemical shift to phospholipid reference standards, and relative class abundances were determined from the corresponding signal integrals.
Preliminary results highlights that 31P NMR has potential to correct for class-specific differences in ionisation efficiency in UHPLC-MSn based lipidomics. However, further optimisation of the 31P NMR workflow is still needed, as some phospholipid signals were broad or overlapping, limiting precise quantification. This could be addressed by testing alternative solvents or using a higher-field instrument. Once a reliable method is established, the same experiment will be applied to different samples from ongoing projects to further validate the approach. Future perspectives will include testing other mass analysers and include triacylglycerols (TGs) by adding standards and correlate it towards one of the phospholipid classes.
References
1 Jurowski K, Kochan K, Walczak J, Barańska M, Piekoszewski W, Buszewski B. Analytical Techniques in Lipidomics: State of the Art. Crit Rev Anal Chem. 2017;47(5):418-437.
2 Züllig T, Trötzmüller M, Köfeler HC. Lipidomics from sample preparation to data analysis: a primer. Anal Bioanal Chem. 2020;412(10):2191-2209.
3 Bril’kov MS, Stenbakk V, Jakubec M, Vasskog T, Kristoffersen T, Cavanagh JP, et al. Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance. Front Mol Biosci. 2023;10:1277963.
4 Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497-509.