Molecular characterization of water extractable Euglena gracilis cellular material composition using asymmetrical flow field-flow fractionation and high-resolution mass spectrometry.

Asymmetrical flow field-flow fractionation (AF4) and high-resolution Orbitrap mass spectrometry (HRMS) were used to separate and characterize cellular fractions of the dark- and light-grown Euglena gracilis cellular material. Biological replicates analyzed by HRMS shared 21-73% of commonly detected m/z values. Greater variability in shared features was found in light-grown cellular fractions (p < 0.05), likely due to small variations in growth stage. Significant differences in molecular composition were observed between AF4 cellular fractions, with dark cell fractions showing a propensity towards carbohydrate-like and tannin-like compounds, and higher double-bond equivalent (DBE) and modified aromatic index (AImod) were associated with light-grown cell fractions. Fractionation and high-resolution mass spectrometry aided characterization demonstrated the power of the AF4 to selectively cater to certain compounds/cellular entities with distinct compositional classes and double-bond equivalents and aromaticity index characteristics. Graphical abstract.