6520 accurate mass q tof

Mammalian CD1a proteins form HEK293 cells were extracted with chloroform, methanol, and water according to a modified Bligh & Dyer method as described^{10 (link)}. The lipid containing organic phase was separated from the aqueous phase and collected for the further analysis. HPLC-MS and lipidomics analysis of CD1a eluted lipids were performed as previously described^{10 (link), 18 (link)}. Briefly, extracted lipids were analyzed with an Agilent Technologies 6520 Accurate-Mass Q-TOF coupled with an Agilent 1200 series HPLC system controlled by MassHunter software. The concentration of the extracted lipids was normalized to the input proteins. Using insect-derived BK6 TCR-CD1aendo complex crystals were separated from mother liquor from approximately 50 crystallisation drops and washed with 6× 400μL crystallisation well solution using a 0.2 μm centrifugal filter at 500g. The harvested crystals were dissolved in 10 mM tris pH 8, 150 mM sodium chloride prior to mass spectrometry as described above.

Optical rotations were obtained on a JASCO P200 polarimeter. Melting points were determined using a Büchi^® B-540 melting point apparatus. IR spectra were measured on a Perkin-Elmer FTIR Spectrum 2. NMR spectra were recorded on a Bruker Avance III - 500 MHz NMR spectrometer equipped with a multi-nucleus probe BBFO (5 mm). UV spectra were recorded on the PDA detector of the Varian LC-920 system. LC-HRESIMS data were recorded on an Agilent 6520 Accurate-Mass Q-TOF hyphenated to an Agilent 1200 system equipped with a Zorbax Agilent C18 column (50 mm × 2.1 mm, 1.8 μm). Compounds were purified by semi-preparative HPLC with a Gilson 322 system equipped with an Axia C18 column (21.2 mm × 100 mm, 5 μm). HPLC analyses were performed on a Varian LC-920 system with a Kinetex column C18 100 Å (100 × 3.0 mm, 2.6 μm). Solid-phase extractions were performed on Chromabond^® SPE cartridges (Macherey-Nagel). The reading of the microplates was performed on an ELISA Versamax plus^® (Molecular Devices) plate reader. Solvents for extraction, fractionation and HPLC were supplied by Sigma-Aldrich. The bioassay material was purchased as following: GLP-1 ELISA kit (Active GLP-1, EGLP-35K Millipore), DMEM and PBS (Gibco), BSA (Sigma-Aldrich) and MTS mother solution CellTiter 96 (Promega), compounds standards pregnenolone and pregnenolone sulphate (Sigma-Aldrich).

Compounds were incubated with 250 nM Rgl2 or its cysteine mutants in buffer (100 mM NaCl, 20 mM Tris pH 8.0, 10 mM MgCl₂, 2 % DMSO) for 24 h at 4 °C. After the incubation, the samples were centrifuged at 20,000 × g for 10 min to remove precipitants prior to being injected into a Zorbax 300-SB C3 column (Agilent, Santa Clara, CA) on an Agilent 1200 liquid chromatography system (Agilent, Santa Clara, CA), using a gradient of Buffer A (H₂O with 0.1 % Formic Acid) and Buffer B (acetonitrile with 0.1 % Formic Acid), and the masses were detected on an Agilent 6520 Accurate Mass Q-TOF.

HPLC-MS was performed on an Agilent 6520 Accurate-Mass Q-TOF using a normal-phase ternary gradient HPLC as previously described.^{E13 (link)} In the fecal lipid extracts, α-galactosylceramide m/z 718.58 was quantified from 4.5 to 5.5 minutes on the basis of the retention time and mass of lipids extracted from B fragilis NCTC 9343. Hexosylceramide m/z 828.69 was identified and sphingomyelin m/z 703.58 was quantified on the basis of mass and retention time of a standard. Ion abundance was quantified by centroid integration as area under the curve (MassHunter, Agilent, Santa Clara, Calif).