Seppack

Frozen spheroid samples were heated for 10 min at 95°C in a lysis buffer containing 4% sodium dodecyl sulfate and sonicated using a probe sonicator. Proteomic sample preparation consisted of reduction and alkylation, methanol chloroform precipitation, and digestion of the proteins by lysyl endopeptidase (LysC, Wako) overnight and trypsin for 6 h. Peptides of the individual samples were labeled with TMTpro 16plex, pooled, and desalted using SepPack (Waters). The peptide mixture was fractionated by high-pH off-line fractionation and concatenated into 8 fractions for phosphopeptide enrichment, which was performed using MagReSyn TiO2 magnetic beads (ReSyn Biosciences). Enriched phosphopeptide samples were analyzed by liquid chromatography (LC) tandem mass spectrometry (MS) on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). A database search of the proteomics data was performed using FragPipe. All statistical comparisons were performed using two-tailed homoscedastic Student t-tests. The mass spectrometry proteomics data files were deposited in the Proteomics Identification Database (PXD036100). Further details are provided in the Supplemental Methods, http://links.lww.com/HEP/I56.

Cell were suspended in PBS and centrifuged at 4000 rpm during 15 min. Pellets were extracted thrice by 2 volumes of CHCl₃/CH₃OH (2/1; v/v) and 1 volume CHCl₃/CH₃OH/H₂O (1/2/0.8; v/v/v). Supernatants were collected and dried gently under N₂ stream. Glycosphingolipids were separated from other lipids and from hydrophilic components on a tC₁₈ cartridge Sep-Pak connected to QMA Sep-pack cartridge (Waters, St Quentin Yvelines, France) equilibrated in CH₃OH/CF₃COOH/H₂O (1/0.1/1; v/v/v) by extensive washing. Neutral glycosphingolipids were eluted in 2 volumes of CH₃OH. Acidic glycosphingolipids were eluted twice in CH₃OH/CH₃COONH₄ (1/1; v/v) within 0.05 M, 0.15 M, and 0.45 M CH₃COONH₄ sequentially in 1 volume each. Neutral and acidic glycosphingolipids were dried gently under N₂ stream. Acidic glycosphingolipids were separated from ammonium acetate on a tC₁₈ cartridges Sep-pack (Waters, St Quentin Yvelines, France) equilibrated in CH₃OH/CF₃COOH/H₂O mixture (1/0.1/1; v/v/v) by extensive washing. Acidic glycosphingolipids were eluted in 2 volumes of CH₃OH and dried under N₂ stream before reconstitution in CHCl₃/CH₃OH (1/2; v/v) and analysis on MALDI-QIT-TOF.

The EMP100 peptide in lyophilised powder form (30, 50, 75, 90 µg vials) and the [^natGa]Ga-EMP100 standard (500 µg vial) were supplied by Edinburgh Molecular Imaging.
ABX (Advanced Biochemical Compounds, Radeberg, Germany) provided all necessary radiolabel materials in a GMP-compliant single-use kit: 0.08 mol/L ammonium acetate buffer, 60% pure ethanol solution, ascorbic acid, 0.9% NaCl saline, water for injection (WFI, BBraun), eluent solution (5 mol/L NaCl, 0. 1 mol/L HCl), cationic SCX (Bond Elut®, Agilent) and C18 reversed-phase columns (Sep-Pack®, Waters), 0.22 µm filter (Millex-GV®, Merck Millipore LTd.) and sterile vials for the final product, with ultrapure gentisic acid provided by Sigma-Merck.
The gallium-68 eluate was obtained by elution of one or two ⁶⁸Ge/⁶⁸Ga generators Galliapharm® (Eckert and Ziegler GmbH, Germany). The automated synthesis of [⁶⁸Ga]Ga-EMP100 was performed on the GAIA synthesis module (Elysia Raytest, Belgium), placed in a high energy class A laminar air flow hot cell MEDI 5000® (Medisystem, France). This module allows the entire process to be edited and controlled by a computer program.