Glass insert

Samples were derivatized for GC-MS analysis according to the method of Kind et al. [16 (link)]. Briefly, 10 µL of methoxyamine hydrochloride dissolved in pyridine (40 mg/mL) was added to each lyophilized sample, and shaken at 30 °C at maximum speed for 90 min using a thermomixer (Eppendorf). A mixture of retention time marker standards were prepared by dissolving fatty acid methyl esters (FAMEs) of different linear chain lengths in chloroform. The FAME mixture (20 µL) was added to 1 mL of N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA) containing 1% trimethylchlorosilane (TMCS), and 90 µL of the FAMEs/MSTFA solution was added to each sample. Samples were shaken at 37 °C at maximum speed in a thermomixer for 30 min, and then transferred to and sealed in amber GC-MS sample vials containing glass inserts (Agilent). Extraction blanks were prepared following the above procedure but starting with empty Eppendorf tubes.

Plasma metabolites’ extraction was made on the basis of a previously described methodology [23 (link)]. In brief, 10 µL of plasma was mixed with 30 µL of cold methanol containing 1 μg/mL of Phe-13C as the internal standard and 1 μM BHT as the antioxidant. After incubation at 4 °C for 60 min, the samples were centrifuged at 12,000× g for 3 min. Then, the supernatant was filtrated (CLS8169, Sigma, Madrid, Spain) and transferred to vials with glass inserts (Agilent, Barcelona, Spain) for chemical analysis. Samples were decoded and randomized before the LC-MS analysis. To serve as quality control for metabolite extraction, a pool of plasma samples with internal Phe-13C was used and injected every 5 samples.

MULTI-BEADS SHOCKER MB1200 (YASUI KIKAI, Osaka, Japan) was used for pulverizing frozen ears. Crushed tissue was dissolved in 1 ml methanol and 500 μl chloroform containing the deuterium-labeled ceramide (1 μM d₉-ceramides containing ester-linked FA, ω-hydroxy FA, and sphingosine (Cer[EOS]) d18:1/32:0/18:2; Cayman, Ann Arbor, MI, USA), followed by incubation at −30°C for 16 h. Samples were centrifuged (2,000 g, 4°C, 10 min), and 200 μl supernatants were dissolved in 100 μl methanol and 20 μl MilliQ water. After incubation (15 min, 20°C), the samples were centrifuged (2,000 g, 20°C, 10 min). The supernatants were applied for untargeted lipidomics.
The frozen epidermis and dermis were dissolved in 50 μl MilliQ water, 50 μl chloroform, and 100 μl methanol. Tissues were then dispersed using scissors. The total samples were mixed with 50 μl chloroform in an internal standard (300 nM d₇-DHS d18:0, d₇-Sph d18:1, d₃-Cer[NS] d18:1/18:0, and d₃-Cer[NDS] d18:0/18:0; Cayman). MilliQ water (50 μl) was added and vortexed. After centrifugation (10,000 g, 20°C, 1 min), the total volume of the precipitate was transferred to a glass insert (Agilent, Santa Clara, CA, USA) in a 2 ml vial. After drying, the dried samples were dissolved in 200 μl methanol. The samples (10 μl) were diluted with 190 μl of methanol and subjected to targeted lipidomics.

The supernatant was transferred to a new 1.5 mL Eppendorf tube and dried using a rotational vacuum concentrator for hormone extraction. The dried extract was reconstituted in 1 mL of 1% AcOH and further purified with a Sep-Pak tC18 cartridge (Waters, United States). The tC18 cartridge was washed by 1 mL methanol, which was followed by an equilibration step with 1 mL 1% AcOH. The sample was loaded into the cartridge, and this was followed with a wash of 1 mL 1% AcOH. The extract was eluted with 1 mL 80% ACN containing 1% AcOH into a new 1.5 mL Eppendorf tube. The eluted extract was dried using a rotational vacuum concentrator and reconstituted in 20 μL 1% AcOH. The extract was vortexed for 30 s until the dried extract dissolved and then was centrifuged at 15,900 rcf at 4°C for 10 min. Samples were transferred to vials with a glass insert (Agilent, United States) and stored at −80°C until UPLC-MS/MS analysis (Figure 1).

Twenty-five micrograms of purified GLA, GBA, and
AGA was dissolved
in 50 mM ammonium bicarbonate (AmBic) buffer (pH 7.4) and further
reduced with 10 mM dithiothreitol (DTT) at 60 °C for 45 min on
a shaker, followed by alkylation with 20 mM iodoacetamide (IAA) at
25 °C for 30 min in the darkness. The sample was proteolytically
digested with chymotrypsin (1:40 enzyme/substrate ratio). The reaction
was quenched with 1 μL of TFA and the digested sample was desalted
by custom-made modified StageTip columns with three layers of C18
and two layers of C8 membrane (3 M Empore disks, Sigma-Aldrich). Samples
were eluted with two steps of 50 μL of 50% methanol in 0.1%
formic acid. The final sample was aliquoted in two equal parts. The
first aliquot was placed into a glass insert (Agilent, USA), dried
completely in a SpeedVac (Eppendorf, Germany), further redissolved
in 50 μL of 0.1% formic acid (FA), and submitted for nLC-MS
analysis. The second aliquot was placed in an Eppendorf tube, dried
completely in a SpeedVac, and redissolved in 50 μL of 50 mM
AmBic buffer (pH 7.4), followed by addition of 1 U PNGase F per sample
at 37 °C for 12 h on a shaker. The sample treated with PNGase
F was desalted, dried by the same methods mentioned above for the
first aliquot, and submitted for nLC-MS/MS analysis.

For polar metabolites analysis, a variation of the method described in Cheong et al. [70 (link)] was used. 250 µL 100% LC-MS grade MeOH (Roth, Karlsruhe, Germany), containing 4% ¹³C₆ sorbitol/valine (Sigma-Aldrich, Castle Hill, Australia) was added to approximately 25 mg of frozen root tissue from each pooled group. Tubes were shaken at 800 rpm for 15 min at 30 °C, centrifuged at 15,700× g for 15 min at room temperature and the supernatant collected. The pellet was re-extracted with 250 µL of Milli-Q H₂O as above and the supernatants combined. In case of cloudy supernatant or precipitate observed during supernatant transfer, the pooled supernatant was centrifuged at 15,700× g for 10 min at room temperature before transferring as much supernatant as possible to a new tube. From each sample, 50 µL aliquots of the supernatant were transferred into glass insert (Agilent, Santa Clara, CA, USA) and dried under vacuum at 30 °C for 90 min.