Orbitrap eclipse tribrid mass

Proteome samples were analysed using LC–MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher) with a FAIMS Pro device using a combination of two compensation voltages of –50 V and –70 V. Chromatographic peptide separation was achieved on 50 cm reverse-phase nanoHPLC-columns (ID 75 μm, PoroShell C18 120, 2.4 μm) coupled to an EASY-nLC 1200 HPLC system and a binary buffer system A (0.1% formic acid) and B (80% acetonitrile/0.1% formic acid). Samples derived from in-solution digestion were measured over a 120 min gradient, raising the content of buffer acetonitrile from 3.2 to 22% over 102 min, from 22 to 45% over 8 min and from 45 to 76% over 2 min. The column was washed with 76% acetonitrile for 8 min. Full MS spectra (300–1,750 m/z) were recorded at a resolution of 60,000, maximum injection time of 20 ms and automatic gain control target of 6 × 10⁵. The 20 most abundant ion peptides in each full MS scan were selected for higher-energy collisional dissociation fragmentation at nominal collisional energy of 30. MS2 spectra were recorded at a resolution of 15,000, a maximum injection time of 22 ms and an automatic gain control target of 1 × 10⁵. This MS acquisition program was alternatively run for both FAIMS compensation voltages to cover different peptide fractions.

The endogenous bound lipids from SPNS2 proteins were extracted by adding chloroform-methanol (2:1, v/v) as previously described.⁴⁸ (link) The extracted lipids were dried and resuspended in 80% methanol. For LC-MS/MS analysis, the lipids were separated on a C18 column (Acclaim PepMap 100, C18, 75 μm × 15 cm, Thermo Fisher Scientific) using a Dionex UltiMate 3000 RSLC nano LC System using a previously described mobile phase and gradient.⁴⁹ (link) The column eluent was loaded to Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). The Orbitrap Eclipse was operated in negative mode using data-dependent acquisition. The detailed mass spectrometer parameters are as follows: spray voltage, -1.9 kV; capillary temperature, 320°C; mass range, 250-1500; full MS resolution, 120K; MS/MS resolution, 15K; NCE, 25/30/35; cycle time, 1.5s. The data was analysed by LipidSearch 4.2 software (Thermo Fisher Scientific).