Evaluation of Extraction Solvents for Untargeted Metabolomics to decipher the Dissolved Organic Matter of Antarctic Cryoconite Holes
Cryoconite holes are biological hotspots with a high biogeochemical turnover rate, contributing significantly to the glacial ecosystem’s overall carbon cycles and net fluxes.
Unfortunately, the information about the composition of low molecular weight molecules formed through the metabolic processes of cryoconite-dwelling microbes is scanty. These molecules constitute a substantial portion of the dissolved organic matter (DOM) within cryoconite holes.
The present study investigated the composition of DOM in cryoconite holes using reverse-phase liquid chromatography (RP-LC) coupled with high-resolution tandem mass spectrometry. We evaluated various solvent combinations of water, methanol, and acetonitrile to extract chemically diverse polar and non-polar metabolites from the cryoconite holes.
Among the single solvents, organic-rich MeOH: Water (70:30 v/v) and in parallel 2-single solvent combinations of MeOH: Water (70:30 v/v) and Acetonitrile: Methanol: Water (40:40:20 v/v) provided increased number and chemical diversity of extracted metabolites.
Combining RP with the hydrophilic interaction liquid chromatography (HILIC) technique provided the highest number of unique metabolites. This dual-LC and ionization polarity combination increased the detection of metabolic features by 46.96% and 24.52% in single- and two-solvent combinations compared to RP alone.
This study developed a simple untargeted metabolomics workflow that is highly sensitive and robust, detecting and potentially identifying a large number of chemically diverse molecules present in the DOM (extracellular) and microbes (intracellular) from the cryoconite holes environment.
This method can better characterize DOM’s chemical composition and, after integrating with other omics approaches, can be used to examine the link between metabolic pathways and microbial communities in global cryoconite holes or other similar ecosystems, revealing how these earthy systems and their microbial flora control carbon or nutrient storage or release in response to global climate change. Overall, the study presents a valuable methodology for studying the biogeochemistry of cryoconite holes.
Overlap of metabolic features detected by HILIC and RP in positive- and negative-ion MS polarities (based on the MS1 neutral mass for the corresponding [M + H] + or [M − H] − ion, +/−0.001 Da). The Venn diagram; (A) feature detected between HILIC best (75HILIC) and RP best (70MW); (B) feature detected between HILIC best (75HILIC) and RP two best solvent combination (70MW and AMW). The stacked bar plot (C) shows unique and shared features between the best HILIC (75HILIC) and RP single solvent (70MW), two solvent combinations. The total number of metabolites detected is displayed on top of each bar. Note that the number of shared metabolites (red) between both techniques is the least in both comparisons. (D) Intensity comparison of the same metabolites detected by HILIC and RP; putatively annotated metabolites with high intensity in RP phase (top row) and metabolic features with high intensity in HILIC phase (bottom row). The HILIC phase detected highly polar metabolic features compared to the RP phase, whereas the RP phase detected moderately polar metabolites. — biorxiv.org
Astrobiology
