Tuning mix

HPLC-purified peptide samples were dried under vacuum and dissolved in 50% CH₃CN with 0.05% formic acid. Mass spectra (ESI-QTOF) were collected on a Bruker maXis and calibrated with Tuning Mix (Agilent). The observed masses were all within 5 p.p.m. of the calculated masses (Supplementary Fig. 11).

The target plates for the API-MALDI/TOF analysis were prepared following the standard procedure previously cited [4 (link)]. Briefly, one volume of the eluted sample was mixed with two volumes of premade matrix solution (6.2 mg of CHCA in 1 mL of 36% methanol, 56% acetonitrile and 8% water). Next, 1 μL of this mixture was spotted in quadruplicate onto the MALDI target plate and allowed to dry at room temperature in a vacuum desiccator. API-MALDI/TOF analysis was performed in positive mode on a 6210 Time of Flight LC/MS (Agilent, Santa Clara, CA, USA) coupled with an atmospheric pressure PDF-MALDI Ion Source (Agilent) equipped with a 337 nm nitrogen laser. The following voltages were applied: fragmentor 300 V, skimmer 60 V, OCT RF 300 V. The acquisition laser power was set at 35% of maximum (peak power 75 kW, pulse energy 300 mJ). Data acquisition was automated using Mass Hunter software (Agilent) and programmed to accumulate 600 shots per spectrum. The irradiation program was automated using the spiral motion control of the PDF-MALDI ion source. The instrument was externally calibrated using Tuning Mix (Agilent). The nominal resolution of the instrument was 20.000 (17.000 observed), and the nominal mass accuracy (with internal calibration) was <2 ppm.

MS grade acetonitrile was obtained from Biosolve (Dieuze, France), formic acid (MS grade) from Acros Organic (Morris Plains, NJ, USA), ethanol from Fisher Chemical (Loughborough, UK), Tuning Mix from Agilent Technologies (Santa Clara, CA, USA), 4-methyl-catechol (4MeC), Amberlyst A-26(OH) ion-exchange resin, periodic acid, 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), citric acid, sodium phosphate dibasic were purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol (99.8%) was purchased from Chemlab. Ultrapure water comes from a Milli-Q system (Merck, Darmstadt, Germany).

ULC-MS grade acetonitrile, ethanol, 2-propanol, methanol and ultra-pure water were purchased from Biosolve (Chimie SARL, Dieuze, France; BV, Valkenswaard, Netherlands). Chloroform was purchased from Carlo Erba (Milan, Italy). NH₄COOH, NH₄F, and butylated hydroxytoluene (BHT) were purchased from Sigma Aldrich (Milan, Italy). The Q-TOF calibration solution was prepared in acetonitrile from Agilent Technologies Tuning mix (HP0321 solution, Agilent Technologies, Santa Clara, USA).
N-palmitoyl-d31-D-erythro-sphingosine (d31-Cer[NS]34:1, MW 569.1), N-[26-oleoyloxy(d9) hexacosanoyl]-d-erythro-sphingosine (Cer[EOS] (d18:1/26:0/18:1(d9), MW 967.6) and N-[26-oleoyloxy hexacosanoyl]-d-erythro-sphingosine (Cer[EOS] (d18:1/26:0/18:1), MW 958.6) were purchased from Avanti Polar Lipids (Alabaster, USA). Deuterated cholesterol sulfate sodium salt (d7-CHS, MW 495.3) and Hexadecanoic-9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-d17 acid (d17-PA, MW 273.5) were purchased from CDN Isotopes Inc., (Pointe-Claire, Canada). Stock solution of the internal standard (iSTD) mixture of deuterated compounds was prepared in 2-propanol with the following concentrations: d7-CHS 40 µM, d17-PA acid 80 µM, d31-Cer16:0 10 µM. d9-CER[EOS] and CER[EOS] mixture (2 µM each in Chloroform/methanol (2:1)) was introduced to confirm identification of O-acylceramides.

The high-resolution mass spectrometry analysis was performed using a SolarixXR 9.4 T (Bruker, Germany), equipped with an electrospray ionization (ESI, Bruker) source, set in negative ionization mode. The instrument was externally calibrated with Tuning Mix from Agilent. Samples were infused directly into the ESI source. The parameters were optimized to obtain a stable ion current with a minima ion injecting time into the mass analyser. The infusion flow rate was 2.0 µL min⁻¹, the drying gas temperature was 200 °C, and the drying and nebulizing gas flow rate were 4.0 L min⁻¹ and 1 bar, respectively. The ESI capillary voltage was 3.9 kV. Three hundred scans were accumulated for each spectrum. Methanol was injected prior to the injection of each sample, and acquisition was performed to evaluate the potential presence of residual pollutants. The acquisition size was set to 8 M, resulting in a mass resolving power of up to (6.6 ± 1.6) × 10⁵ for the full mass range.

The methanol extract of N. biserrata was analyzed using an Agilent 1290 Infinity LC system coupled with an Agilent 6520 QTOF mass spectrometry system. A 2.0 µl sample was injected into a reversed-phase column, namely Agilent Zorbax Eclipse XDB-C18 (narrow bore, 2.1 mm × 150 mm × 3.5 µm; Agilent Technologies, Santa Clara, CA, USA). The column was maintained at 25 °C at a flow rate of 500µL/min during analysis. The mobile phases were composed of solvent A (H₂O—0.1% HCOOH) and solvent B (acetonitrile—0.1% HCOOH). The gradient elution program was initiated at 5% of solvent B for 5 min, then from 5 to 100% solvent B in 15 min. and kept for 5 min. Later, the column was conditioned as initial for 5 min. prior to next injection.
The mass spectrometry (MS) signals were acquired as described by Ling et al. (2018)^{38 (link)} with minor modifications. Briefly, voltage for positive electrospray ionization was changed to 4 kV, while the remaining settings for ion source were maintained. The MS was calibrated with Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) before each batch analysis. Internal mass calibration standards, purine (m/z 121.0508) and hexakis-(1H, 1H, 3H-tetrafluoropropoxy)-phosphazine (m/z 922.0097), were introduced throughout the runs for automated mass correction^{39 (link)}.