Speedvac

Brain samples were freeze-dried (FreeZone® 4.5 L, Labconco, USA) at −107 °C and 0.2 mbar for 12 h. The lyophilized material (3 mg) was mixed with 750 µL of ACN/H₂O (1:1), vortexed for 15 s, homogenized by gentle planar shaking at 4 °C for 30 min and centrifuged at 11,500 × g and 4 °C for 15 min (Thermo Fischer, USA). Supernatants (500 µL) were concentrated under vacuum (SpeedVac, Thermo Fischer, USA) at 45 °C for 4 h. The resultant dry residues were reconstituted in 100 µL of MeOH/H₂O (1:9).
Urine samples were diluted to the tenth with ultrapure water and vortexed for 15 s.
Metabolites were extracted from 100 µL of plasma by adding 800 µL of MeOH/H₂O (1:1). The samples were then vortexed for 15 s, homogenized by gentle planar shaking at 4 °C for 30 min, and centrifugated at 11,500 × g and 4 °C for 10 min to collect 500 µL of supernatant. Supernatants were concentrated under vacuum (SpeedVac, Thermo Fischer, USA) at 45 °C for 4 h. The dried residues were reconstituted in 100 µL of MeOH/H₂O (1:9). Each prepared sample was then transferred to a 96-well plate for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Quality control samples (QCs) were obtained from a pooled mixture of equal volumes of all samples for each matrix. Fifteen QCs were injected to equilibrate the chromatographic system before each analytic batch and QCs were analyzed every 10 samples.

Cerebral organoids (COs) were washed with PBS, treated for 1 h with cell recovery solution (CRS, Corning, NY, USA) at 4 °C, and washed with PBS again. CO was homogenized in a microtube with a glass bead (Benchmark Scientific, Edison, NJ, USA). The COs were stored at -80 °C before processing. COs were freeze-dried (Savant SDP121 P, SpeedVac, ThermoFisher Scientific, USA) and homogenized using a glass bead. Lipid, ganglioside, and metabolite extraction were performed by adding 100 µl of 80% isopropanol to the homogenate. It was followed by a brief vortex, sonication (37 Hz, 5 min), and vortexing (200 rpm, 10 min). The extract was then centrifuged (12.3 RCF, 5 min), and the filtrate was removed and mixed 1:1 with a mixture of lipid and metabolite internal standards (Supplementary Table 2) for lipidomics, and metabolite assays or with a mixture of isotopically labeled GM1 and GM3 internal standards for ganglioside assay, and stored in -20 °C until LC-MS analysis. The protein pellet was dried using the SpeedVac vacuum concentrator (Savant SDP121 P, ThermoFisher Scientific). The dried protein pellet was reconstituted to perform the BCA assay protein determination. Control (N = 10) and tramiprosate-treated COs (N = 10) were utilized for the selective reaction monitoring - mass spectrometry (SRM-MS) analysis. The protein pellet was dried to be processed for the proteomics assay.

For fecal samples, 20 mg feces and 50 mg glass beads (Sigma-Aldrich, MO) were added to 400 µL cooled methanol solution (methanol: water 1: 1), followed by homogenizing using a TissueLyser (QIAGEN) for 15 min at 50 Hz. The supernatant was collected after centrifuging for 10 min at 1,2000 rpm, dried up in a SpeedVac (Thermo Scientific), and then resuspended for metabolomic profiling. For plasma samples, 80 µL cooled methanol was added to 20 µL plasma. After incubation for 30 min at −20 °C, the samples were centrifuged for 10 min at 1,2000 rpm. The supernatant was collected, dried up in a SpeedVac (Thermo Scientific), and then resuspended.

For cells lysis, 50 μL 8 M urea was added directly to each well and cells were scratched off, transferred, and subsequently sonicated for 1 h at 4°C. Afterwards 200 μL of 0.8% RapiGest (Waters) within 50 mM NH₄HCO₃ and 2 mM DTT were added and incubated for 1 h at room temperature (RT), followed by addition of 10 mM MMTS (Pierce, Erembodegem, Belgium) and incubation of 1 h at RT in the dark. Digestion of proteins in peptides was achieved by addition of 1 μg Trypsin Gold (Promega) for 24 h at RT and stopped by addition of 1% TFA for 1 h at RT. Afterwards samples were salted out by using 3 M Empore cartridges (3 M Bioanalytical, MS). Samples were loaded on cartridges, eluted with 200 μL of 70% acetonitrile (ACN) within 0.1% TFA, and further dried in vacuum centrifuge (Speedvac, Thermo Scientific). Dried samples were resuspended with 20 μL of 0.1% TFA. Samples were loaded on Zip Tips C18 (ZTC18S960, Millipore, Molsheim, France) and eluted with 20 μL 70% ACN within 0.1% TFA and dried in vacuum centrifuge (Speedvac, Thermo Scientific). Before measurement, samples were resuspended in 10 μL of 2% ACN within 0.1% TFA.

For DXM, a 100 μL plasma sample was vortex mixed with 600 μL methanol for 5 min. After that, the mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was pipetted to a clean glass tube and eluted in a phospholipid removal tube (Phree Phospholipid removal; Phenomenex, Torrence, CA, USA). The eluate from the phospholipid removal tube was collected in a clean glass tube and dried in a vacuum evaporator (SpeedVac, Thermo Fisher Scientific, Auckland, New Zealand). The LCMS water (100 μL) was used to reconstitute the dried residue, and 10 μL of the mixture was injected into the LCMS column.
For butorphanol, a 100 μL plasma sample and 400 μL methanol was vortexed mixed for 10 min. Then, the mixture was centrifuged at 12,500 rpm for 10 min. The supernatant was transferred to a phospholipid removal tube (Phree Phospholipid removal; Phenomenex, Torrence, CA, USA). The eluate from the phospholipid removal tube was collected in a clean glass tube and dried in a vacuum evaporator (SpeedVac, Thermo Fisher scientific, Auckland, New Zealand). The methanol (100 μL) was used to reconstitute the dried residue, and the mixture was centrifuged at 12,000 rpm for 5 min. After centrifuging, 10 μL of the mixture was injected into the LCMS column.

Fecal samples were freeze-dried (FreeZone^®4.5 L, Labconco, Kansan City, MO, USA) at −107 °C, 0.2 mbar for 24 h and then pooled and mixed as previously described by Martias et al. [28 (link)].
For each extraction protocol, the extraction was repeated 10 times. Five extraction procedures were tested on 50 mg for each of a pool of dried feces: H₂O:MeOH:CHCl₃ (1:1:1), ACN:H₂O (1:1), MeOH:H₂O (1:1), MeOH:H₂O (4:1), MeOH:H₂O:ACN (1:1:1). Samples were vortexed during 10 min and centrifuged (10 min at 4 °C, 15,000 g). Supernatants were collected in 2 aliquots (one for the NMR analysis and the other for MS analysis) for further solvent evaporation in a SpeedVac (ThermoFisher). A graphical visualization of all sample preparation protocols is given in Figure S1c.
For the MS analysis, dried-residues were dissolved in 150 µL of ACN:H₂O (4:1). 75 µL were used for HILIC and the remaining phase were evaporated in a SpeedVac (ThermoFisher). Then, dried-residues was dissolved in 75µL of MeOH:H₂O (1:9) for RP-LC. For the ¹H-NMR analysis, dried-residues were dissolved in 200 μL of a deuterated buffer (0.2 M potassium phosphate buffered deuterium oxide (pH = 7.44 ± 0.5) and 10 μL of deuterium oxide (D₂O) with external reference [3-trimethylsilylpropionic acid (TSP) at 3.2 mM]).