Exploring sex differences in zebrafish livers using a novel targeted discovery metabolomics approach

Targeted workflows using triple quadrupole (QQQ) instruments have recently gained more attention in the field of metabolomics. The targeted aspect, namely having to decide which metabolites to measure prior to data acquisition, is a barrier for untargeted metabolomics. However, we have shown previously that even a QQQ instrument can be used to acquire data with the same level of metabolite coverage as conventional untargeted metabolomics experiments. As new nominal mass instruments with high acquisition rates, sensitivity, and advanced features like MSn emerge, our goal is to expand the boundaries of what can be achieved through a targeted approach, and propose an alternative to untargeted analysis. Using a targeted discovery metabolomics experiment, we measured liver extracts from male and female zebrafish.

Livers from adult male and female zebrafish (Danio rerio) were harvested. Polar metabolites were extracted with 2:2:1 acetonitrile/methanol/water (40 µL per mg wet weight). A pooled sample was prepared using an aliquot of all samples. Untargeted metabolomics data were acquired using hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer. After feature detection and grouping using Compound Discoverer 3.4, a list of identified and unidentified compounds was generated and used as a precursor list for the new hybrid nominal mass instrument. Data were acquired in parallel reaction monitoring (PRM) mode using fast polarity switching and stepped collision energies.

Our data revealed significant sex-based differences in metabolite levels: over 30% of the compounds had a p-value < 0.05 after Benjamini-Hochberg correction and a fold change (|FC|) ≥ 1.5. Threonine levels were higher in female livers, uridine 5'-diphosphoglucuronic acid was seven times higher in females, and methylhistidine was twice as high in males. Additionally, using the instrument"s MSn fragmentation capability and an MS3 method, we characterized the fatty acid composition of triglycerides. Most lipids were increased in female livers, except for those containing fatty acid 18:2, which were lower compared to males.