TITLE: Glucosylceramide and glucosylsphingosine analysis
AUTHORS: Tae-Un Han, Carley Corado

[DESCRIPTION]
This protocol was used to analysis glucosylceramide and glucosylsphingosine levels in mouse brain and liver.
It was also described in previous publication (Mol Cell Neurosci. 2020 Jan:102:103451, doi: 10.1016)

[STEPS]
SECTION: Tissue preparation
1. The mouse brain and liver tissues (100 - 300 mg) were homogenized in 2% CHAPS solution (4 mL/g wet tissue) in 2 mL Omni homogenization tubes containing 8 mm ceramic beads 
SECTION: Tissue preparation
2. The homogenates were processed on the Bead Ruptor 24 (Omni International, Kennesaw, GA) for two 30 second cycles at 5.65 m/s with a 45 second pause time. 
SECTION: liquid chromatography-tandem mass spectrometry
3. The glucosylsphingosine and glucosylceramide in homogenate (50 μL) were extracted and analyzed
SECTION: liquid chromatography-tandem mass spectrometry
4. The brain internal standards including d5-GluCer(18:0) (250 ng/mL) for GluCer and d5-GluSph
(200 ng/mL) for GluSph were prepared in acetonitrile solution.
SECTION: liquid chromatography-tandem mass spectrometry
5.  The samples were vortexed for approximately 3 min and then centrifuged at 10,000 rpm for 10 min. The supernatants were transferred to 1.2 mL glass inserts (VWR, West Chester, PA) in 96 well plates.
SECTION: liquid chromatography-tandem mass spectrometry
6. A quality control (QC) sample was prepared by pooling 20% of extracts from study samples and used to monitor the instrument performance.
SECTION: liquid chromatography-tandem mass spectrometry
7. The crude extracts were directly injected to LC–MS/MS system for analysis of GluSph and GluCer. CSF and GluSph in brain homogenates and the injection volumes were 100 μL and 5 μL, respectively.
SECTION: liquid chromatography-tandem mass spectrometry
8. LC–MS/MS analysis was conducted on a Shimadzu (Columbia, MD) Prominence HPLC system coupled with an Applied Biosystems/MDS Sciex (Ontario, Canada) 4000QTRAP mass spectrometer using multiple reaction monitoring (MRM)
SECTION: liquid chromatography-tandem mass spectrometry
9. The HPLC system consists of Prominence HPLC system with a CBM-20A system controller, 2 LC-20 CE pumps, a SIL-20ACHT autosampler, and a DGU-20A5R degasser. The compartment of the autosampler was set at 4 °C.
SECTION: liquid chromatography-tandem mass spectrometry
10. For analysis of GluSph, the chromatography was performed at ambient temperature using Ascentis‱ Express HILIC (4.6 × 50 mm, 2.7 μm, Supelco, Bellefonte, PA) protected with a HILIC Securityguard‱ column (4 × 3.0 mm, Phenomenex, Torrance, CA)
SECTION: liquid chromatography-tandem mass spectrometry
11. The compartment of the autosampler was set at 4 oC.
SECTION: liquid chromatography-tandem mass spectrometry
12. Mobile phase A (0.1% formic acid and 1 mM ammonium formate in water) and mobile phase B (0.1% formic acid and 1 mM ammonium formate in acetonitrile-water (95:5)) were operated with a gradient elution as follows: 0–0.2 min 100–95% B, 0.2–3.5 min 95% B, 3.5–3.9 min 95–90% B, 3.9–5.8 min 90% B, 5.8–5.9 min 90–10% B, 5.9–6.9 min 10% B, 6.9–7.0 min 10–100% B, and 7.0–8.8 min 100% B at a flow rate of 1.5 mL/min.
SECTION: liquid chromatography-tandem mass spectrometry
13. The HPLC flow was diverted to waste except for 2.0–6.5 min to mass spectrometer.
SECTION: liquid chromatography-tandem mass spectrometry
14. The ESI source temperature was 600 °C; the ESI needle was 5000 V; the declustering potentials for
GluSph and d5-GluSph were 76, 76, 76, 76, and 80 V, respectively; both the entrance potential and the collision cell exit potential were 10 V for all compounds. 
SECTION: liquid chromatography-tandem mass spectrometry
15. The collision and curtain gas were set at medium and 20, respectively.
SECTION: liquid chromatography-tandem mass spectrometry
16. Both desolvation gas and nebulizing gas were set at 45. For MRM, the collision energies
for mass transitions of m/z 462.3 to 282.3 for GluSph and that for d5-GluSph were 31, 26, 38, and 31 V, respectively.
SECTION: liquid chromatography-tandem mass spectrometry
17. The dwell time was set at 50 ms for each mass transition
SECTION: liquid chromatography-tandem mass spectrometry
18. For analysis of GluCer,  the chromatography was performed at ambient temperature using Supelcosil‱ LC-Si (2.1 × 250 mm, 5 μm, Supelco, Bellefonte, PA) protected with a HILIC Securityguard‱ column (4 × 3.0 mm, Phenomenex, Torrance, CA).
SECTION: liquid chromatography-tandem mass spectrometry
19. Mobile phase A (5 mM ammonium acetate in water) and mobile phase B (5 mM ammonium acetate in acetonitrilemethanol-acetic acid (97:2:1)) were operated with a gradient elution as follows: 0–4.0 min 100% B, 4.0–4.1 min 100–95% B, 4.1–6.5 min 95% B, 6.5–6.6 min 95–10% B, 6.6–7.6 min 10% B, 7.6–7.7 min 10–100% B, 6.9–7.0 min 10–100% B, and 7.0–10 min 100% B at a flow rate of 1.5 mL/min. 
SECTION: liquid chromatography-tandem mass spectrometry
20. The HPLC flow was diverted to waste except for 2.0–6.5 min to mass spectrometer
SECTION: liquid chromatography-tandem mass spectrometry
21. The ESI source temperature was 550 °C; the ESI needle was 5000 V; the declustering potentials for
GluCer, GalCer, and DihexCer were 75, 75, and 80 V, respectively; both the entrance potential and the collision cell exit potential was 10 V for all the compounds.
SECTION: liquid chromatography-tandem mass spectrometry
22. The collision and curtain gas were set at medium and 20, respectively.
SECTION: liquid chromatography-tandem mass spectrometry
23. The desolvation gas and nebulizing gas were set at 35 and 55, respectively.
SECTION: liquid chromatography-tandem mass spectrometry
24. For MRM, the collision energies for mass transitions of m/z 700.5 to 264.3 for GalCer(16:0) and GluCer(16:0), m/z 728.5 to 264.3 for GalCer(18:0) and GluCer(18:0), m/z 756.5 to 264.3
for GalCer(20:0) and GluCer(20:0), m/z 784.6 to 264.3 for GalCer(22:0)
and GluCer(22:0), m/z 812.7 to 264.3 for GalCer(24:0) and GluCer
(24:0), m/z 810.7 to 264.3 for GalCer(24:1) and GluCer(24:1), m/z
862.7 to 264.3 for DihexCer(16:0), m/z 890.7 to 264.3 for DihexCer
(18:0), m/z 918.7 to 264.3 for DihexCer(20:0), m/z 946.7 to 264.3 for
DihexCer(22:0), m/z 974.8 to 264.3 for DihexCer(24:0), m/z 972.7 to
264.3 for DihexCer(24:1), m/z 733.5 to 269.3 for d5-GluCer(18:0), and
m/z 865.7 to 264.3 for d3- LacCer(16:0) were 50, 50, 50, 50, 50, 50, 61,
67, 67, 67, 67, 50 and 61 V, respectively. 
SECTION: liquid chromatography-tandem mass spectrometry
25. The dwell time was set at 50 ms for each mass transition.
SECTION: liquid chromatography-tandem mass spectrometry
26. Data processing was conducted with Analyst 1.5.1 (Applied Biosystems).
SECTION: liquid chromatography-tandem mass spectrometry
27. The signal of noise ratio of analyte < 3 was defined as below limit of detection (LOD).
SECTION: liquid chromatography-tandem mass spectrometry
28. The relative quantification data were obtained as peak area ratios of analytes to their internal standards, which were converted to concentrations.