A stable-isotope mass spectrometry-based metabolic footprinting approach to analyze exudates from phytoplankton

Abstract

Phytoplankton exudates play an important role in pelagic ecology and biogeochemical cycles of elements. Exuded compounds fuel the microbial food web and often encompass bioactive secondary metabolites like sex pheromones, allelochemicals, antibiotics, or feeding attractants that mediate biological interactions. Despite this importance, little is known about the bioactive compounds present in phytoplankton exudates. We report a stable-isotope metabolic footprinting method to characterise exudates from aquatic autotrophs. Exudates from (13)C-enriched alga were concentrated by solid phase extraction and analysed by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. We used the harmful algal bloom forming dinoflagellate Alexandrium tamarense to prove the method. An algorithm was developed to automatically pinpoint just those metabolites with highly (13)C-enriched isotope signatures, allowing us to discover algal exudates from the complex seawater background. The stable-isotope pattern (SIP) of the detected metabolites then allowed for more accurate assignment to an empirical formula, a critical first step in their identification. This automated workflow provides an effective way to explore the chemical nature of the solutes exuded from phytoplankton cells and will facilitate the discovery of novel dissolved bioactive compounds.

Figure 1

Flow scheme for the stable isotope metabolic footprinting approach for marine microalgae. Algal cells are cultured in either ¹²C (control with natural isotopic distribution) or ¹³C-enriched media. Cells are removed by filtration and metabolites in the cell free filtrate concentrated onto solid phase extraction columns. Eluted compounds are then analyzed using FT-ICR mass spectrometry and a novel algorithm is used to automatically locate the stable isotope patterns, compare them to theoretical isotope intensity profiles, and output the empirical formula(e) of the exuded metabolite(s).

Figure 2

Representative region of mass spectra (m/z 370–412) from negative ion direct infusion (DI) FT-ICR metabolic footprinting analyses across four different sample groups: (a,b) unlabelled and ¹³C-labelled seawater without algal cells, respectively, as controls, and (c,d) unlabelled and ¹³C-labelled cultures of Alexandrium tamarense, respectively; all according to the workflow described in Figure 1. The bottom panel shows several prominent isotope patterns arising from ¹³C-labelled exudates that are absent from all other spectra, indicating successful incorporation of the stable isotope into A. tamarense’s biochemical pathways and subsequent transfer to the culture media.

Figure 3

Principal component analysis scores plot from analysis of the DI FT-ICR mass spectra (m/z 70–590) from a metabolic footprinting study of () unlabelled and ¹³C-labelled () seawater without algal cells (as controls, n = 6 each), and () unlabelled and () ¹³C-labelled cultures of Alexandrium tamarense (n = 6 each). The major separation along the PC1 axis corresponds to the differences between the metabolic footprints of seawater samples with versus without algal cells present. Separation along PC2 corresponds to differences between the metabolic footprints of ¹³C-labelled vs. unlabelled A. tamarense cultures.

Figure 4

(a,c,e) A representative subset of the stable isotope patterns used for estimating the labelling efficiency observed in DI FT-ICR mass spectra collected from ¹³C-labelled cultures of Alexandrium tamarense. The “signal intensity template” (see Methods) is presented in green, additional ¹³C-labelled peaks in black, and isotopic peaks observed in mass spectra of unlabelled A. tamarense samples in red. Based on accurate mass measurements and the number of peaks, each SIP was annotated with a single formula: [C₇H₁₀O₂ − H]⁻, [C₆H₁₂O₃ − H]⁻ and [C₁₄H₂₈O₂ − H]⁻ for the three plots, respectively; (b,d,f) Simulated stable isotope patterns (SIPs) modelled for each empirical formula applying five different labelling efficiencies: 50% (green), 55% (blue), 60% (red), 65% (orange) and 70% (yellow). SIPs are shown as continuous data to avoid the presentation of overlapping peak intensities; the experimental data for ¹³C-labelled A. tamarense, identical to that in the plots on the left, are shown in grey, the average labelling efficiency was estimated to be ~58% (see Table 1, including the Pearson’s correlation coefficients (r) and associated p-values for each comparison between an experimental and simulated SIP).

Figure 5

(a,c,e). Stable isotope patterns observed in DI FT-ICR mass spectra collected from ¹³C-labelled cultures of Alexandrium tamarense. Signal intensity template presented in green, additional ¹³C-labelled peaks in black, and isotopic peaks observed in mass spectra of unlabelled A. tamarense samples in red. (b,d,f). Simulated stable isotope patterns (SIPs) were modelled for each empirical formula assignment to the all-¹²C peak with a labelling efficiency of 58%, where the coloured profiles correspond to differing numbers of carbon atoms in the formula (defined in Table 2). The experimental data for ¹³C-labelled A. tamarense, identical to that in the plots on the left, are shown in grey. The results of the metabolite annotation are shown in Table 2.