Ultrahigh-Resolution Mass Spectrometry Method for Resolving ¹³C-Enrichment Patterns in a Microalgal Lipidome

Abstract

The analysis of 13 C-labeled lipids by mass spectrometry is challenging due to the complexity from labeling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionization and in-source fragmentation, which can create complex overlapping isotope patterns that can only be resolved using high-resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13 C-labeled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labeling information. Mass resolving powers of 240000 full width half-maximum (fwhm) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts, and unresolved lipid species after chromatographic separation. This enabled the percentage of 13 C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labeling within the headgroup vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and remodeling processes and can be extended to other biological systems.

Recurrent Topics in Mass Spectrometry-Based Metabolomics and Lipidomics—Standardization, Coverage, and Throughput

“…The list of labeled organisms is further growing. For example, uniformly 13 C labeled lipids derived from microalgae Nannochloropsis oculata were measured via MS/MS to calculate 13 C enrichment for both the whole molecule and the different building blocks of a lipid . Such information can be useful to follow labeling of the head group versus fatty acids and might help to study lipid synthesis and remodeling processes.…”

“…For example, uniformly 13 C labeled lipids derived from microalgae Nannochloropsis oculata were measured via MS/MS to calculate 13 C enrichment for both the whole molecule and the different building blocks of a lipid. 60 Such information can be useful to follow labeling of the head group versus fatty acids and might help to study lipid synthesis and remodeling processes. Advances in stable isotope labeling in plants using customized closed growth chambers enabled us to increase the enrichment degree to 96−98% for 13 C and 95−99% for 15 N adding a complex compound panel of primary and secondary metabolites.…”