Tsp d4

A total of 15 μL of 5 mM 3-(Trimethylsilyl-propionic-2,2,3,3-d₄) acid (TSP-d₄; Sigma-Aldrich, Oakville, ON, Canada) in 100% deuterium oxide (D₂O; Sigma-Aldrich) was added to the rotor as an internal standard set to 0.0 ppm prior to the addition of the insert. Analysis was performed in a Bruker 600 MHz NMR spectrometer (Billerica, MA, USA) equipped with an advanced HR-MAS probe using the water suppression pulse sequence, zgpr (Bruker standard sequence). Sixty-four scans were acquired, with an acquisition time of 0.97 s and a spectral width of 8.4 kHz.

The analysis of metabolites was performed using the protocol as described in our previous study [20 (link)] with suitable modification. Approximately 20 mg of each sample was weighted and extracted with acetonitrile/water (1:1, v/v) mixture on ice for 10 min. The samples were centrifuged at 3,000×g for 10 min at 4°C, and the supernatant was collected and freeze-dried. The lyophilized samples were dissolved in 700 μL of deuterated water containing 2 mM 3-trimethylsilyl-2,2,3,3-tetradeuteropropionicacid-d4 (TSP-d4; Sigma-Aldrich, St. Louis, MO, USA) as an internal reference, and then was transferred into a 5 mm NMR tube for analysis. ¹H-NMR spectra were acquired on a 600 MHz Agilent NMR spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with 600 MHz 4-mm gHX NanoProbe (Agilent Technologies, Santa Clara, CA, USA) at a ¹H frequency of 599.93 MHz. The ¹H-NMR conditions set were the same as those described in our previous study [20 (link)]. The acquired spectra were phased and then the baseline was corrected and referenced to the TSP-d4 peak using a Vnmrj (version 4.2, Agilent Technologies, USA). Metabolites in the ¹H-NMR spectra were tentatively identified using Chenomx 600 MHz library database and Chenomx NMR Suite 7.1 professional (Chenomx Inc., Edmonton, Canada). The identified metabolites were expressed as percentage of the normalized area.

¹H-NMR spectra of 10 and 600 µM of D-sphingosine in PBS (D-erythro-Sphingosine, Matreya, Cat# 1802) were recorded at 15 °C in 90% deuterium oxide (D₂O, Sigma-Adlrich, Cat# 151882) and 10% DMSO-d₆ using trimethylsilylpropanoic acid (TSP-d₄, Sigma-Aldrich, Cat# 269913) as an internal standard. ¹H NMR spectra of 5, 10, 200, 600, and 2400 µM of psychosine were recorded at 15 °C in 90% D₂O and 5% DMSO-d₆ using TSP-d₄ as an internal standard. Data were processed and analyzed with the Topspin 3.8 or 4.0.1 NMR software.

All reagents and solvents used were of analytical grade and they were purchased from Mallinckrodt Chemical Works (St. Louis, MO, USA), Alfa Aesar GmbH & Co (Karlsruhe, Germany) and Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). NMR solvents and standards D₂O (99.9%), NaN3 (99.5%) and TSP-d4 (97%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin–Ciocalteu’s phenol reagent and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were obtained from Sigma Chemical Co. (St. Louis, MO, USA), ABTS [2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] from Tokyo Chemical Industry Co. Ltd (Tokyo, Japan) and 3,4,5-trihydroxybenzoic acid from Alfa Aesar (Karlsruh, Germany).

1D¹H-NMR spectra of 600 µM dopamine (Dopamine hydrochloride, Sigma-Aldrich, Cat# H8502) in PBS were recorded at 15 °C in 10% deuterium oxide (D₂O, Sigma-Adlrich, Cat# 151882) using trimethylsilylpropanoic acid (TSP-d₄, Sigma-Aldrich, Cat# 269913) as an internal standard. 1D ¹H NMR spectra were recorded at 0.5, 24, 48, 72, 96, and 105 hours. The sample was stored at 4 °C between each time point to match the same conditions of dopamine and psychosine in presence of α-synuclein for 15 and 96 hours. As we did not notice any dopamine oxidation products, samples were kept open to air at r.t and monitored for any color change as a sign of oxidation. On day 16, an intense change in color and a precipitate were observed. The precipitate was separated and reconstituted in 100% DMSO-d₆ (Sigma-Aldrich, Cat# 151874) and 1D ¹H NMR spectra were recorded at 15 °C. Data were processed and analyzed with the Topspin 3.8 or 4.0.1 NMR software.