Xcalibur software version 4

Each peptide sample was dissolved in formic acid (FA, 10%) and 5 µL was injected into a nano-ACQUITY UPLC system (Waters, Milford, MA, USA). Peptides were separated on a 1.7 mm BEH C₁₈ column (Waters, Milford, MA, USA) at a flow rate of 300 nL/min. Peptide elution was achieved with a linear gradient (solution A: H₂O (95%), CH₃CN (5%), FA (0.1%); solution B: CH₃CN (95%), H₂O (5%), FA (0.1%)); 15–50% B over 180 min). MS and MS/MS data were acquired with an LTQ-Orbitrap XL (ThermoFisher, Waltham, MA, USA). The fifteen most intense doubly and triply charged peptide ions were chosen by the Xcalibur software version 4.0 (ThermoFisher, Waltham, MA, USA.) and fragmented. The resulting MS data were processed to generate peak lists for protein identifications.

The lipids were analyzed by LC-MS/MS using a Thermo Vanquish HPLC (Thermo Fisher Scientific, Germering, Germany). MS was performed with heated ESI source in positive and negative mode, respectively. The spray voltage was set to 3.5 kV for positive and −2.8 kV for negative mode, and ion transfer capillary was 325°C. Nitrogen was used as both sheath gas and auxiliary gas and was set to 35 and 15 arbitrary units, respectively, and the auxiliary gas temperature was 250°C. For MS/MS, higher-energy collision dissociation (HCD) with nitrogen gas and step collision energy (NCE) of 20, 30, 50 for positive mode, and 20, 30, and 50 for negative mode were used to present a broader range of fragment ions and collected as much informative data as possible. MS data were acquired in the scan range of m/z 80–1200 and were processed using Xcalibur software version 4.0 (Thermo Scientific, San Jose, CA, United States).

The resulting peptides were analysed by online nanoliquid chromatography coupled to an MS/MS instrument (Ultimate 3000 RSLCnano and Q-Exactive HF, Thermo Fisher Scientific) using a 360-min gradient for proteome-wide analysis and a 200-min gradient for interactome characterization. For this, peptides were sampled on a 300 μm × 5 mm PepMap C18 precolumn and separated in a 200 cm µPAC column (PharmaFluidics) or a 75 μm × 250 mm C18 column (Aurora Generation 2, 1.7 µm, IonOpticks) for, respectively, proteome-wide and interactome analyses. MS and MS/MS data were acquired using Xcalibur software version 4.0 (Thermo Scientific).

The chromatographic system (Dionex UltiMate 3000 UHPLC system, Dionex Softron GmbH, Germering, Germany) consisted of a binary pump (HPG-3400RS), an autosampler (WPS-3000RS), a degasser (SRD-3400), and a column oven (TCC-3000RS). Detection was carried out on a quadrupole/orbital ion trap Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Analytes were separated on a reversed-phase Ascentis Express C18 column (2.1 mm × 100 mm, 2.7 µm) from Supelco (Bellefonte, PA, USA). The LC-MS system was equipped with a heated electrospray ionization source (HESI-II) and Xcalibur software, version 4.0 (Thermo Fisher Scientific, San Jose, CA, USA).

The dried peptides were dissolved in 5% ACN with 0.1% FA immediately before analysis and loaded onto a 150 mm x 75 µm PepMap RSLC capillary analytical C18 column with 2 μm particle size (LC Packings) using Ultimate 3000 HPLC system (Dionex, Idstein, Germany). Peptide separation was done at a flow rate of 0.300 µl/min, and the solvents gradient started at 3 min and was ramped to 60% Buffer B (90% ACN + 0.03% FA) over 60 min. Eluted peptides were analyzed using a Q Exactive HF mass spectrometer with a nanoelectrospray ionization source (ESI) (Thermo Fisher Scientific, Bremen, Germany). The mass spectrometer was externally calibrated and operated in positive and data-dependent modes. Survey MS scans were conducted in the 200–2000 m/z range, with a resolution of 120,000. The top ten most intense ions were fragmented with 30 eV collision energy on an HCD collision cell and analyzed with a resolution of 30,000. A 10 s dynamic exclusion window was applied, and an isolation window of 4 m/z was used to collect suitable tandem mass spectra. The obtained data were acquired with the Xcalibur software version 4.1 (Thermo Fisher Scientific, Bremen, Germany).