Acetonitrile

Acetonitrile, chloroform, methanol, acetic acid, phosphoric acid, and hydrochloric acid were purchased from Carl Roth GmbH & Co KG (Karlsruhe, Germany). Ammonium acetate, copper sulfate, and potassium chloride were purchased from Sigma-Aldrich GmbH (Munich, Germany). The standard substances SQDG 816 (2-O-hexadecanoyl-1-O-(9Z,12Z,15Z-octadecatrienoyl)glycerol 3-(6-deoxy-6-sulfo-α-d-glucopyranoside)) and 3-O-sulfo-d-galactosyl-β1-1′-N-heptadecanoyl-d-erythro-sphingosine as internal standard (ISD) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All aqueous solutions were prepared with deionized water, generated by a Purelab flex water purification system (Veolia Water Technologies Deutschland GmbH, Celle, Germany). NH₂ cartridges (6 mL, 500 mg) were purchased from Macherey-Nagel GmbH & Co. KG (Düren, Germany).

The galactolipid standards were purchased from Avanti polar Lipids Inc. (Alabaster, AL, USA). Acetonitrile, chloroform, and methanol were purchased from Carl Roth GmbH and Co KG (Karlsruhe, Germany). Ammonium acetate, copper sulfate, and potassium chloride were obtained from Sigma-Aldrich GmbH (Munich, Germany). Additionally, ammonia (25%) and nitric acid (65%) were acquired from Merck KGaA (Darmstadt, Germany). All aqueous solutions were prepared with deionized water, generated by a Purelab flex water purification system (VeoliaWater Technologies Deutschland GmbH, Celle, Germany). Amino-phase (NH₂) solid phase extraction cartridges (6 mL, 500 mg) were purchased from Macherey-Nagel GmbH and Co. KG (Düren, Germany).

Monobutyl phthalate (MBP, 99% purity), di-n-butyl phthalate (DBP, 99%), diethylhexyl phthalate (DEHP, 99.5%), diethyl phthalate (DEP, 99%), dimethyl phthalate (DMP, 99%), phthalic acid (PA, > 99.5%), 2-ethyl-1-hexanol (99.6%), terephthalic acid (98%), and isophthalic acid (99%) were purchased from Sigma Aldrich Chemie GmbH (Hamburg, Germany). Acetonitrile, ethyl acetate, hexane, and formic acid were of HPLC grade and purchased from Carl Roth (Karlsruhe, Germany).

The reagents were supplied from Alfa Aesar (Haverhill, MA, USA) or Sigma Aldrich (Saint Louis, MO, USA). Organic solvents (acetonitrile, DMF, chloroform, and methanol) were supplied from POCh (Polskie Odczynniki Chemiczne, Gliwice, Poland). All chemicals were of analytical grade. Before use, dried DMF and acetonitrile were kept in crown cap bottles over anhydrous phosphorus pentoxide (Carl Roth, Karlsruhe, Germany).
The NMR spectra were recorded on a Bruker AVANCE DMX400 (Bruker, Billerica, MA, USA) spectrometer, operating at 500 or 300 MHz (¹H NMR) and 125 or 75 MHz (¹³C NMR). The chemical shift values are expressed in ppm relative to TMS as an internal standard. Mass spectral ESI measurements were carried out on Waters ZQ Micro-mass instruments (Waters, Milford, MA, USA) with a quadrupole mass analyzer. The spectra were performed in the positive ion mode at a declustering potential of 40–60 V. The sample was previously separated on a UPLC column (C18) using the UPLC ACQUITYTM system by Waters connected with a DPA detector. Flash chromatography was performed on Merck silica gel 60 (200–400 mesh) using chloroform/methanol (19:1 vol) mixture as eluent. Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm thickness).

First, 1.6 µg (4 µL) of ANXA1 and PNGaseF-deglycosylated antibody, respectively, was analyzed by LC-MS using a UHPLC system (UltiMate 3000, RSLCnano, Dionex GmbH, Idstein, Germany) coupled to a QTOF-type mass spectrometer (MaXis HD UHR q-TOF, Bruker, Bremen, Germany). Reversed-phase chromatography (ACQUITY BEH C4, 1.7 µm, 300 Å, 1 mm × 50 mm, Waters GmbH, Eschborn, Germany) was applied for desalting. The solvents were 0.1% formic acid in (A) water (Carl Roth, Karlsruhe, Germany) and in (B) acetonitrile (Carl Roth, Karlsruhe, Germany), respectively. A stepwise linear gradient was run at 75 °C at a flow rate of 150 µL/min (0–0.4 min, 5% B; 2.5 min 30% B; 7 min, 50%B) followed by wash steps. Mass spectrometer parameters were adapted to the size of the molecule and the LC flow rate.

Alkane C₈-C₂₀ analytical standard (40 mg/L), 2,2-diphenyl-picrylhydrazyl (DPPH), methanol (HPLC grade), rutin (95%), and trifluoroacetic acid (99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, USA); acetonitrile (>99%) and hexametylentetramine (99%) were supplied by Carl Roth (Karlsruhe, Germany); acetic acid (glacial) and aluminum chloride (99.17%) were purchased from Chempur (Piekary Śląskie, Poland); Folin-Ciocalteu phenol reagent and n-heptane (HPLC grade) were purchased from Merck (Darmstadt, Germany); sodium acetate (98%) and sodium carbonate (99.5%) were supplied by Reachem (Petržalka, Slovakia); carbon dioxide (99.9%) and helium (99.999%) gases were purchased from AGA (Vilnius, Lithuania); microcrystalline cellulose was purchased from Chempol (Židlochovice, Czech Republic); and the xanthohumol analytical standard (≥98%) was kindly supplied by Axen Bio (Gdańsk, Poland).

Fmoc-L-phenylalanine, Fmoc-L-glutamic acid 5-tert-butyl ester, and N-α-Fmoc-N-ß-trityl-L-asparagine were procured from Fluka (Buchs, Switzerland), while Fmoc-O-tert-butyl-L-tyrosine was sourced from Roth (Karlsruhe, Germany). Furthermore, Rink amide resin (4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, PEPTIPURE^®, 100–200 mesh, 0.3–0.8 mmol·g⁻¹, 1% DVB) purchased also from Roth was used as a solid support. Various chemical reagents essential for experimental procedures were obtained from reputable suppliers. Dimethyl sulfoxide (DMSO, EMSURE ACS), diethyl ether (EMSURE ACS), N,N-dimethylformamide (DMF), piperidine (PYP), triisopropylsilane (TIS), acetic acid, benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate (PyBOP), N-methylmorpholine (NMM), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (HCCA), and NaH₂PO₄ were acquired from Merck (Darmstadt, Germany). Dichloromethane (DCM) was sourced from Scharlab (Barcelona, Spain), while methanol, acetonitrile (ACN, HPLC grade), and trifluoroacetic acid (TFA) were obtained from Carl Roth (Karlsruhe, Germany). All solutions were prepared meticulously using water of high purity with a resistivity of 18.2 MΩ∙cm, generated through a Millipore Milli-Q system (Bedford, MA, USA).