Rslcnano hplc

Samples from desalting were resuspended in 5% formic acid and 1 μg of peptides was used for analysis. A Dionex RSLCnano HPLC was used for the peptide chromatography. A 5 mm PepMap-C18 pre-column with an inner diameter of 0.3 mm was used and a 75 μm × 50 cm PepMap-C18 column was used for the subsequent chromatography. The mobile phase consisted of 2% acetonitrile + 0.1% formic acid (solvent A) and 80% acetonitrile + 0.1% formic acid (solvent B). A constant flow rate of 300 nL/min was used and the linear gradient increased from 5% to 35% solvent B over a runtime of 156 minutes. The eluted peptides were injected into a Velos Orbitrap mass spectrometer (Thermo) through a nanoelectrospray emitter. A typical ‘Top15’ acquisition method was used. The primary mass spectrometry scan (MS1) was performed at a resolution of 60,000. The aforementioned top 15 most abundant m/z signals from the MS1 scan were selected for subjected for collision-induced dissociation and MS2 analysis in the Orbitrap mass analyzer at a resolution of 17,500.

HPLC-ESI-MS/MS analyses of the LV samples were conducted on a Thermo Fisher Q Exactive mass spectrometer with online separation using either a Thermo Fisher/Dionex RSLC nano HPLC (for lipids) or an Ultimate 3000 HPLC (for polar metabolites).

In all samples, urushiol was extracted using a single phase Folch procedure⁴² and analyzed using a Q-Exactive Hybrid Quadrupole-Orbitrap. Prior to urushiol extraction, crystals of CD1a-urushiol were extensively washed with the crystallization mother liquor and dissolved in Tris Buffer Saline (TBS), pH 8.0. MS coupled to a RSLC nano HPLC (Thermo Scientific, Bremen, Germany). Samples were loaded onto a nanoviper pepmap100 trap column (100μm × 2cm) in 2% acetonitrile / 0.1% ammonium acetate at a flow rate of 15 μL / minute. Analytes were separated at a flow rate of 300 μL / minute on a pepmap100 C18 column (75μm × 15cm, Thermo Scientific) using a linear gradient of acetonitrile (2-80%). Up to 12 MS/MS spectra were acquired per cycle with maximum accumulation time of 50 ms and 100 ms for MS1 and MS2, respectively. A SCIEX QTRAP 5500 mass spectrometer was used for MRM-based detection as previously described⁴³ . The 5500 mass spectrometer was operated with unit quadrupole resolution and three MRM transitions were simultaneously monitored in detecting U15 315.2→149.1, 315.2→135.1, 315.2→122.1. Data analysis was performed using a combination of Analyst v1.5.2 and XCalibar 3.0 (Thermo Fisher Scientific).