Polar lipidomic profile shows Chlorococcum amblystomatis as a promising source of value-added lipids

Total lipid content and fatty acid composition of Chlorococcum amblystomatis

The lipid extracts of Chlorococcum amblystomatis corresponded to an average yield of 20.77 ± 0.57% of microalgal biomass. The fatty acid composition of Chlorococcum amblystomatis identified by GC–MS showed that C16:0 was the most abundant FA (23.0 ± 0.8%), followed by C18:3 n-3 (19.4 ± 0.6%), C18:0 (13.9 ± 2.3%), C16:4 n-3 (11.1 ± 0.6%), C20:5 n-3 (8.9 ± 0.6%) and C16:1 n-9 (7.5 ± 0.3%) (Table 1). The remaining identified FAs had relative abundances of less than 5%. This microalga had a total omega-3 fatty acid content of 43.2%.

To assess the potential health benefits of Chlorococcum amblystomatis, the atherogenic (AI), thrombogenic (TI), hypocholesterolemic/hypercholesterolemic (h/H) and the PUFA n-6/ PUFA n-3 indices were calculated. The AI and TI of the lipid extracts were 0.5 and 0.3, respectively, while the h/H ratio was 1.4 and the n-6/n-3 ratio was 0.1.

Polar lipid composition of Chlorococcum amblystomatis

The polar lipid profile was revealed by hydrophilic interaction liquid chromatography-high resolution electrospray mass spectrometry (HILIC–HR–ESI–MS and HILIC–HR–ESI–MS/MS) (Supplementary file S1). Using this approach, a total of 245 molecular ions, with a minimum of 350 molecular species, belonging to 15 classes of lipids were identified. Of these, 101 molecular ions were glycolipids (GL), 54 molecular ions were betaine lipids (BL), 89 molecular ions were phospholipids (PL), and 1 molecular ion was an inositol phosphoceramide (PI-Cer) (Tables 2 and 3). Figures explaining how the MS/MS data were interpreted can be found in the Supplementary Figures S1–S13. The m/z of the fragment ions used to identify each molecular ion and each molecular species can be found in Supplementary Table S1 and S2.

The PL classes identified included phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE), identified in the HILIC–HR–ESI–MS analysis as the molecular ion [M+H]⁺ (Fig. 1); and phosphatidylglycerol (PG) and phosphatidylinositol (PI) identified as the negative molecular ion, [M−H]⁻ (Fig. 2). The only sphingolipid species found in Chlorococcum amblystomatis was a PI-Cer, which has been identified in the analysis on the negative ion mode. A total of 32 molecular ions of PC, 11 of LPC, 13 of PE, 9 of LPE, 22 of PG, 2 of PI and 1 of PI-Cer were recognized. Analysis of the MS/MS data allowed to recognize that each ion can correspond to several molecular species, with different combinations of fatty acyl chains, but with the same (C:N) (Supplementary Table S2). The molecular species identified per class were of 39 PC, 11 LPC, 14 PE, 9 LPE, 32 PG, 2 PI, and 1 PI-Cer.

As previously indicated, the identification of the lipid species was based on the mass accuracy, retention time and interpretation of MS/MS data. The interpretation of the MS/MS data of the molecular ions of the PC, LPC, PE and LPE species allowed to confirm their identity (Supplementary Table S2). This was done by confirming for each species the presence of the polar head groups, in particular by the presence of the product ion at m/z 184 (for PC and LPC) and fragment ions arising from the neutral loss of 141 Da (for PE and LPE)^16,44–48. On the other hand, the complementary analysis of the molecular ions on the HILIC–HR–ESI–MS/MS spectra acquired in negative ion mode, observed as [M+CH₃COO]⁻ ions for PC and LPC and [M−H]⁻ ions for PE and LPE, allowed to assign the fatty acyl composition. This was done by observing the presence of the RCOO⁻carboxylate fragment anions of the fatty acids esterified in the glycerol backbone^16,44–48.

The HILIC–HR–ESI–MS/MS spectra of the negative molecular ions belonging to the PG, PI and PI-Cer classes allowed to confirm the presence of the corresponding polar heads by observing for each species the product ions at m/z 171 (for PG) and m/z 241 (for PI and PI-Cer), and identify the fatty acyl chains by the presence of RCOO⁻carboxylate anions (for PG and PI)^16,44–48 (Supplementary Table S2). The only PI-Cer detected was identified by the accurate mass measurement, the retention time, and the presence of the polar head at m/z 241. The most abundant species in each phospholipid class were: PC(34:2) identified as PC(16:1–18:1) and PC(16:0–18:2); LPC(16:1); PE(34:4) identified as PE(16:0–18:4); LPE(16:0); PG(34:3) identified as PG(16:0–18:3); and PI(32:1) identified as PI(16:0–16:1).

Among the PG molecular ions identified, a few oxidized PG species were found (Table 3), the most abundant being PG(34:2-OH), identified as PG(18:0-OH-16:2). All the oxidized PG species were identified based on exact mass measurements, retention time and MS/MS spectra analysis (Supplementary Table S3). In the MS/MS spectra, the presence of mass shifts of + 16 Da relative to the [M−H]⁻ ions and of RCOO⁻carboxylate hydroxyl anions was indicative of the formation of hydroxy derivatives^49,50. No product ions with oxidized polar head groups were observed ⁴⁵.

The betaine lipids identified in the HILIC–HR–ESI–MS data included the classes of monoacylglycerol-trimethylhomoserine (MGTS) and diacylglycerol-trimethylhomoserine (DGTS), and were identified in positive ion mode as [M+H]⁺ ions (Table 4, Fig. 3). A total of 38 molecular ions of DGTS and 16 molecular ions of MGTS were identified, corresponding to 68 molecular species of DGTS and 16 molecular species of MGTS. In the HILIC–HR–ESI–MS/MS spectra of MGTS and DGTS, as [M+H]⁺ ions, the presence of the polar head group in each species was confirmed by observation of the product ion at m/z 236^44,47, and the fatty acyl composition was determined by observation of the fragment ions resulting from the neutral losses of the fatty acyl chains as an acid (–RCOOH) and ketene (–R=C=O) derivatives^44,47 (Supplementary Table S2).

The most abundant ions in each class of betaine lipids were MGTS (18:4) and DGTS (34:4) assigned as DGTS (18:4–16:0) and DGTS (18:3–16:1) (Table 4). Interestingly, among the identified DGTS molecular ions, oxidized DGTS molecular ions were also found, the most abundant being DGTS (34:7-OH), identified as a combination of two molecular species of DGTS (16:4-OH-18:3) and DGTS (16:3-OH-18:4). These oxidized ions were identified based on exact mass measurements, retention times, and by analysis of MS/MS spectra (Supplementary Table S3). The MS/MS of the oxidized DGTS molecular ions showed a neutral loss of H₂O and the product ions formed due to the loss of oxidized fatty acyl chains (acid and keto derivatives). Also, the product ion characteristic of betaine lipids at m/z 236 was present in all MS/MS spectra, therefore no product ions with oxidized polar head groups were observed.

The glycolipids identified in the Chlorococcum amblystomatis samples included monogalactosyldiacylglycerol (MGDG), monogalactosylmonoacylglycerol (MGMG), digalactosyldiacylglycerol (DGDG) and digalactosylmonoacylglycerol (DGMG), identified as [M+NH₄]⁺ ions (Fig. 4, Table 5). We also identified sulfoquinovosyl diacylglycerol lipids (SQDG) and a sulfoquinovosyl monoacylglycerol lipid (SQMG), identified as [M−H]⁻ ions (Fig. 5, Table 5). Analysis of the HILIC–HR–ESI–MS and MS/MS spectra identified the following number of molecular ions: 30 MGDG, 11 MGMG, 27 DGDG, 11 DGMG, 21 SQDG and 1 SQMG (Supplementary Table S2). From these molecular ions, we identified the following number of molecular species using MS/MS data: 47 MGDG, 11 MGMG, 48 DGDG, 11 DGMG, 40 SQDG and 1 SQMG.

The typical fragmentation observed in the HILIC–HR–ESI–MS/MS spectra of neutral glycolipids, as [M+NH₄]⁺ ions, allowed to confirm the polar head group by the neutral loss of 197 Da (for MGDG) or 359 Da (for DGDG), which corresponds to the loss of the carbohydrate moiety combined with the loss of NH₃^44,47. The assignment of the fatty acyl composition was corroborated by the presence of product ions corresponding to each fatty acyl group as acylium plus 74 [RCO+74]⁺ ion^44,47 (Supplementary Table S2). On the other hand, the HILIC–HR–ESI–MS/MS spectra of the [M−H]⁻ ions of the acidic glycolipids (SQDG and SQMG), when available, showed the product ion of the sulfoquinovosyl polar head group at m/z 225 and the fatty acyl composition was confirmed by the neutral loss of fatty acyl chains as carboxylic acid (-RCOOH) and by the presence of carboxylate RCOO⁻ anions (Supplementary Table S2).

The most abundant species in each class of glycolipids were: MGDG (34:7), identified as MGDG (16:4–18:3) and MGDG (18:4–16:3); MGMG (16:4); DGDG (34:3), identified as DGDG (16:0–18:3) and DGDG (18:1–16:2); DGMG (18:3); and SQDG (32:1), identified as SQDG (16:0–16:1). The only identified glycolipid species belonging to the SQMG class was identified by accurate mass measurements and by the presence on the MS/MS spectrum of the product ion corresponding to the polar head at m/z 225. Among the MGMG and SQDG species identified, we observed three oxidized species, MGMG (16:4-OH), SQDG (34:3-OH) identified as SQDG (18:3-OH-16:0) and SQDG (34:4-OH) identified as SQDG (18:4-OH-16:0). HILIC–HR–ESI–MS/MS analysis of all oxidized GL species suggested that a hydroxyl group was present in the fatty acyl group (Supplementary Table S3).

The relative quantification of the identified species was carried out as described in the methods section. Figure 6 shows the most abundant lipid species, identified as molecular ions, in the polar lipidome of Chlorococcum amblystomatis, with a relative amount greater than or equal to 1.5%. Glycolipids were the group of lipids with the largest number of species (101). The most abundant species was MGDG (34:7), assigned as MGDG (16:3–18:4) and MGDG (16:4–18:3), and the second most abundant species was SQDG (16:0–16:1).

LC–MS data was acquired using an internal standard for each class of phospholipids. LC–MS data were normalized against their assigned internal standard, to semi-quantify each molecular ion. Due to the lack of commercially available GL and BL standards, internal standards from classes with a retention time closer to those identified for GL and BL were used to normalize their respective data, as performed in our laboratory^45,52–54. The use of one internal standard is acceptable for semi-quantification because the ionization efficiency of polar lipids depends mainly on the polar head, while the length of the chain and the degree of unsaturation contribute little^55,56. However, we would like to point out the limitations of our inter-class quantitation approach, because despite the similar retention time, the response factors for each class, even between phospholipids, tend to differ in ESI–MS, which prevents a robust and accurate quantification^51–53. Normalized data were then used to semi-quantify the abundances of the identified molecular ions. For a clearer overview of the composition of the polar lipidome of Chlorococcum amblystomatis, we have summed up the relative percentage of all the detected lipid species belonging to glycolipids, betaines and phospholipids (Fig. 7A). According to the largest number of molecular ions, the highest cumulative levels were observed for glycolipids (51.9%), followed by betaine lipids (30.1%) and phospholipids (17.9%). The sum of the relative percentage of all lipid species belonging to each class of lipids was also calculated. The highest cumulative levels were observed for DGTS (27.6%) followed by SQDG (23.7%) (Fig. 7B).

Evaluation of in vitro anti-inflammatory and antioxidant properties of Chlorococcum amblystomatis lipid extracts

The anti-inflammatory potential of lipid extracts from Chlorococcum amblystomatis were evaluated using a test kit for screening for inhibition of human COX-2. With 10 µl of an extract of 50 µg mL⁻¹ (5 µg), we have observed an inhibition of 87.5 ± 0.1 of COX-2 activity (Fig. 8). These results suggest that the lipid extracts of Chlorococcum amblystomatis have anti-inflammatory potential.

The antioxidant potential of lipid extracts from Chlorococcum amblystomatis were evaluated using free radical DPPH^• and ABTS^•+ scavenging assays (Fig. 9), as described in the methods section. The results obtained for the DPPH assay revealed that the concentration of the extract resulting in a 40% inhibition (IC40) of DPPH^• was at an estimated concentration of 226.81 ± 2.99 µg mL⁻¹. The average antioxidant activity (Trolox equivalent, TE) was 76.25 ± 1.02 Trolox µmol g⁻¹ of lipid extract. In contrast, the lipid extract which resulted in a 50% inhibition (IC50) of ABTS^•+ was 37.69 ± 4.44 µg mL⁻¹ and the expressed TE activity of 428.81 ± 54.32 Trolox µmol g⁻¹ of lipid extract.

Reagents

HPLC grade methanol (MeOH), ethanol absolute, and dichloromethane (CH₂Cl₂), were purchased from Fisher Scientific Ltd. (Loughborough, UK). All other reagents were purchased from major commercial sources. Milli-Q water (Synergy, Millipore Corporation, Billerica, MA, USA) was used. Phospholipid internal standards 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (dMPE), 1,2-dimyristoyl-sn-glycero-3-phospho-(10-rac-glycerol) (dMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (dMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol (dPPI), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), according to the methodology previously described^34,35. DPPH^• was purchased from Aldrich (Milwaukee, WI). 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS^•+) was obtained from Fluka (Buchs, Switzerland). The 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich (St Louis, MO, USA). The cyclooxygenase (COX-2) inhibitory screening assay was performed using a commercial kit, Cayman test kit-701080 from Cayman Chemical Company (Ann Arbor, MI, USA). All the other reagents and chemicals used were of the highest grade of purity commercially available.

Microalgae material

The spray-dried biomass of Chlorococcum amblystomatis was supplied by Allmicroalgae, Natural products S.A. located in Pataias, Portugal, Fábrica Cibra Pataias, 2445-287 Pataias. The strain was isolated locally and is deposited in the Allmicroalgae culture collection under the code 0066CA ³⁴.

Portugal Chlorococcum amblystomatis 0066 CA was cultivated autotrophically in Guillard’s F2 culture medium, whose composition was adapted to local water, using nitrates as the source of nitrogen ⁹¹. Briefly, 5 L flask reactors were cultivated from 7 to 15 days, under continuous exposure to light. Five 5 L flask reactors were used to inoculate one 0.1 m³ L outdoor Flat Panel (FP) reactor, which was later sequentially scaled as follows: 0.25 m³ L to a 0.5 m³ L to a 1 m³ Flat Panels. Four of the later reactors were used as inoculum of a 10 m³ photobioreactor (PBR). The reactor was operated for 21 days, exposed to the environmental light and temperature conditions, at an average temperature of 15.5 °C and light irradiance of 20.10 MJ m⁻² day⁻¹. pH was maintained constant, 7.0–8.0, by pulse injections of CO₂ and the temperature was kept under 28 °C by a sprinkler-like irrigation system. After growing period, the biomass was industrially collected by centrifugation and further spray-drying.

Lipid extraction procedure

Lipid extraction was carried out using a mixture of dichloromethane: methanol solvents (DM) (2:1, v/v). The lipids were extracted from 25 mg of lyophilized Chlorococcum amblystomatis biomass using the solvent mixture. The suspension was centrifuged (Selecta JP Mixtasel, Abrera, Barcelona, Spain) at 2000 rpm for 10 min and the supernatant was collected in a new pre-weighed glass tube. This process was repeated four times until the extraction solvent lost the green colour. The combined supernatants were dried under a stream of nitrogen.

The Folch extraction method was used to the obtained dried supernatants^54,92. The extracts were redissolved in 2 mL of dichloromethane, and 1 mL of methanol and 0.75 mL of Milli-Q water were added. The mixture was vortexed for 2 min followed by phase separation by centrifugation at 2000 rpm for 10 min. The organic phase was collected in a new pre-weighed tube, and the aqueous phase was reextracted with 2 mL of dichloromethane, two more times. The combined organic phases were dried under a stream of nitrogen and weighted.

Each series of extracts was repeated five times and the total lipid content was determined by gravimetry. The yield of lipids extracted from dry biomass extracts (DW) was calculated as follows (Eq. 1):

COX-2 inhibition assay

A commercial cyclooxygenase (COX-2) inhibitory screening assay kit—Cayman test kit-701080 (Cayman Chemical Company, Ann Arbor, MI, USA)—was used to assess their anti-inflammatory potential ⁹³. This screening assay directly measures the amount of prostaglandin F2α generated from arachidonic acid (AA, C20:4 [n-6]) in the cyclooxygenase reaction. This assay was carried out according to the instructions described by the supplier of the assay kit, using an aliquot of test extract or DMSO. For this assay, lipid extracts of Chlorococcum amblystomatis (500 μg, 250 μg, 125 μg and 50 μg) were dissolved in DMSO and the final volume of reaction was 1000 μl. Positive and negative controls were provided by the assay kit protocol. The positive control used inactivated COX-2 enzyme, and negative control used the enzyme with 100% initial activity without any inhibitor. The assay was performed in three replicates. Interferences were considered by subtracting COX-2 inhibition from the blank assays. The results were expressed as a percentage of inhibited COX-2.

DPPH Radical Scavenging Assay

The antioxidant scavenging activity against the α,α-diphenyl-β-picrylhydrazyl radical (DPPH^•) was evaluated as described previously^94,95 with some modifications. Briefly, 150 µL of an ethanolic dilution of the extracts (25, 125, 250, 500 µg mL⁻¹) or 150 µL of the Trolox standard solution (5, 12.5, 25, 37.5 µmol L⁻¹ in ethanol) were mixed in triplicate with 150 µL of a DPPH^• working solution in ethanol (absorbance ~ 0.9, 517 nm). The mixture was incubated for 120 min and the absorbance was measured at 517 nm every 5 min (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA). A control was prepared by replacing the DPPH^• solution with ethanol. The antioxidant activity, expressed as a percentage of inhibition of the DPPH radical, was calculated using the following equation (Eq. 2):

The activity is expressed in Trolox Equivalents, which were calculated using Eq. 2, where IC40 values are the concentration of sample or of Trolox that induces the reduction the DPPH• radical to 40%) (Eq. 3):

ABTS radical cation scavenging assay

The antioxidant scavenging activity against the 2,20-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS^•+) was evaluated using a method previously described^94,96 with some modifications. Briefly, 150 µL of an ethanolic dilution of the extracts (25, 125, 250, 500 µg mL⁻¹) or 150 µL of the Trolox standard solution (5, 12.5, 25, 37.5 µmol L⁻¹ in ethanol) were mixed in triplicate with 150 µL of an ABTS^•+ working solution in ethanol (absorbance ~ 0.9, 734 nm). The mixture was incubated for 120 min and the absorbance was measured at 734 nm every 5 min (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA). A control was prepared by replacing the ABTS^•+ solution with ethanol. The antioxidant activity, expressed as a percentage of inhibition of the ABTS radical (IC40 substituted by IC50), was calculated using Eq. (2) (Abs_DPPH^• substituted by Abs_ABTS^•+) and expressed in Trolox Equivalents (Eq. 3).

Analysis of fatty acids by gas chromatography–mass spectrometry (GC–MS)

The fatty acid methyl esters (FAMEs) were prepared from total lipid extracts of Chlorococcum amblystomatis by transmethylation reaction using a methanolic solution of potassium hydroxide (2.0 M) according to the methodology previously described ⁹⁰. A volume of 2.0 μL of a hexane solution containing FAMEs and 1.03 μg mL⁻¹ of methyl nonadecanoate (Sigma, St. Louis, MO, USA) as internal standard was injected in a chromatography-mass spectrometry (GC–MS) (Agilent Technologies 8860 GC System, Santa Clara, CA, USA) equipped with a DB-FFAP column with the following specifications: 30 m long, 0.32 mm internal diameter, and 0.25 μm film thickness (J & W Scientific, Folsom, CA, USA). The GC equipment was connected to an Agilent 5977B Mass Selective Detector operating with electron impact ionization at 70 eV and a scanning range of m/z 50–550 (1 s cycle in a full scan mode). The following conditions were used: helium as carrier gas (constant flow 1.4 mL min⁻¹), inlet temperature 220 °C, detector temperature 230 °C, injection volume 2 μL (splitless). The oven temperature was programmed as follows: 58 °C for 2 min, 25 °C min⁻¹ to 160 °C, 2 °C min⁻¹ to 210 °C, 30 °C min⁻¹ to 225 °C (held for 20 min). The data acquisition software used was GCMS5977B/Enhanced MassHunter. Data were analysed using Agilent MassHunter Qualitative Analysis 10.0 software. The identification of FA was carried out by comparison of the MS spectrum with the NIST chemical database library and confirmed with the literature reports. Five independent replicates were injected.

Polar lipidome analysis by hydrophilic interaction liquid chromatography coupled to high-resolution tandem mass spectrometry (HILIC–HR–MS/MS)

The polar lipidome was determined according to the methodology previously described^34,35. The dried samples were dissolved in CH₂Cl₂ to a final concentration of 1 ug uL^-1. From each sample, a volume of 10 µL (10 µg of lipid extract) was taken and transferred to an appropriate vial, followed by the addition of 86 µL of a solvent system composed of two mobile phases in a proportion of 90% v/v eluent B and 10% v/v eluent A and 4 µL of a mixture of internal standards (dMPC—0.02 μg, dMPE—0.02 μg, SM (17:0/d18:1)—0.02 μg, LPC—0.02 μg, dPPI—0.08 μg, dMPG—0.012 μg, dMPS—0.04 μg). The composition of eluent B was 60% v/v of acetronitrile, 40% v/v of methanol, and 5 mM of ammonium acetate and the composition of eluent A was 50% v/v of acetonitrile, 25% v/v of methanol, 25% v/v of water, and 5 mM ammonium acetate.

The lipids were separated by hydrophilic interaction liquid chromatography (HILIC) using a microbore Ascentis Si column (10 cm × 1.0 mm, 3 µm; Sigma-Aldrich) and a high performance-liquid chromatography (HPLC) system (Ultimate 3000 Dionex, Thermo Fisher Scientific, Bremen, Germany) with an autosampler coupled to the Q-Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A 5 µL aliquot of each sample mixture was injected into the HPLC column, at a flow rate of 50 µL min⁻¹ and a temperature of 35 °C. The following gradient was applied: 10% A (0–2 min), 10–90% A (2–15 min), 90% A (15–17 min). The mass spectrometer with Orbitrap technology was operated simultaneously in positive (electrospray voltage 3.0 kV) and negative (electrospray voltage − 2.7 kV) modes, with the following configuration: high resolution with 70,000, AGC target of 1 × 10⁶, capillary temperature 250 °C, and sheath gas flow of 15 U. The tandem mass spectrometry experiments were performed according to the following configuration: resolution of 17,500, AGC target of 1 × 10⁵, with one full scan mass spectrum and 10 data-dependent MS/MS scans. The cycles were repeated continuously throughout the experiments with the dynamic exclusion of 60 s and an intensity threshold of 1 × 10⁴. Normalized collision energy (CE) ranged between 25, 30, and 35 eV. Data acquisition was performed using the Xcalibur data system (V3.3, Thermo Fisher Scientific, USA). Five independent biological replicas were carried out.

Data analysis

To identify the classes of the polar lipids in the lipid extracts acquired spectra were analysed using Xcalibur v3.3 (Thermo Fisher Scientific, USA). The identification was carried out according to a standard approach in our laboratory ⁹⁸: this approach consists of the localization of the species according to the retention time of internal standards (information related to internal standards can be found in Supplementary Table S1), accurate mass measurements (5 ppm) and identification of recurring fragmentation patterns (Supplementary Figures S1–S13) and their comparison with those of internal standards and published information on fragmentation patterns^98–101. After identification, the quantification of molecular species was carried out by integrating the chromatographic peaks using the MZmine v2.42 software. The software allows filtering and smoothing, peak detection, peak processing and assignment against an internal database ¹⁰². All peaks of raw intensity bellow 1 × 10⁴ were excluded.

Relative quantification was performed by exporting the values of the peak areas to a computer spreadsheet (Excel, Microsoft, Redmond, WA). To normalize the data, the peak areas of the extracted ion chromatograms (EIC) of each lipid molecular species were divided by the EIC peak areas of the selected internal standards. Relative abundances were calculated using dMPC, dMPE, SM(17:0/d18:1), LPC, dPPI, dMPG and dMPS as internal standards. To normalize DGTS and MGTS we used PE as internal standard, for SQDG we used PG as internal standard, and for MGDG, DGDG, DGMG and MGMG, we used Ceramide as internal standard. For clear visualization, normalized data was transformed in percentage. The normalized data was calculated as follows (Eq. 7):

The relative percentage of betaines, phospholipids and glycolipids was calculated as follows (Eq. 8):

The relative percentage of the distribution of omega-3 and omega-6 fatty acids across the different main classes of polar lipids (betaines, phospholipids and glycolipids) was calculated as follows (Eq. 9):

Raw abundances, normalized data and relative quantification can be found on the additional spreadsheet (Supplementary file S1).