Orbitrap lumos

All samples (~ 1 µg for each fraction) were loaded and eluted by an Easy-nLC 1200 (Thermo Scientific) with an in-house packed 15 cm, 150 μm i.d. capillary column with 1.7 μm CSH (Water’s) over a 35 min gradient. Mass spectrometry was performed with a high-field asymmetric waveform ion mobility spectrometry (FAIMS) Pro front-end equipped Orbitrap Lumos (Thermo) in positive ion mode using data-dependent acquisition with 1 s top speed cycles for each FAIMS compensative voltage. Each cycle consisted of one full MS scan followed by as many MS/MS events that could fit within the given 1 s cycle time limit. MS scans were collected at a resolution of 120,000 (410–1600 m/z range, 4 × 10^5 AGC, 50 ms maximum ion injection time, FAIMS compensative voltage of -45 and -65). Only precursors with charge states between 2 + and 5 + were selected for MS/MS. All higher energy collision-induced dissociation (HCD) MS/MS spectra were acquired at a resolution of 50,000 (0.7 m/z isolation width, 35% collision energy, 1 × 10^5 AGC target, 86 ms maximum ion time). Dynamic exclusion was set to exclude previously sequenced peaks for 30 s within a 10-ppm isolation window.

We used nanoflow LC-MS/MS using Orbitrap Lumos (Thermo Fisher Scientific) coupled to an Agilent 1290 HPLC system (Agilent Technologies). Trap column of 20 mm × 100 μm inner diameter (ReproSil C18, Dr Maisch GmbH, Ammerbuch, Germany) was used followed by a 40 cm × 50 μm inner diameter analytical column (ReproSil Pur C18-AQ (Dr Maisch GmbH, Ammerbuch, Germany)). Both columns were packed in-house. Trapping was done at 5 μl/min in 0.1 M acetic acid in H₂O for 10 min, and the analytical separation was done at 300 nl/min for 2 h by increasing the concentration of 0.1 M acetic acid in 80% acetonitrile (v/v). The mass spectrometer was operated in a data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra were acquired in the Orbitrap from m/z 350–1500 with a resolution of 60,000 FHMW, automatic gain control (AGC) target of 200,000 and maximum injection time of 50 ms. Ten most intense precursors at a threshold above 5000 were selected with an isolation window of 1.2 Da after accumulation to a target value of 30,000 (maximum injection time was 115 ms). Fragmentation was carried out using higher-energy collisional dissociation (HCD) with collision energy of 38% and activation time of 0.1 ms. Fragment ion analysis was performed on Orbitrap with resolution of 60,000 FHMW and a low mass cut-off setting of 120 m/z.

Metabolome analyses were performed on a Thermo Ultimate RS 3000 coupled online to a Q-Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization probe (HESI II). The MS was calibrated by Thermo calmix (Pierce™ calibration solutions in both polarities. Lipidome analysis was performed on an Ultimate RS 3000 UHPLC (Thermo Fisher), coupled online to a TimsTOF Pro Quadrupole Time of Flight (Q-TOF) (Bruker Daltonics, Bremen, Germany) equipped with an Apollo II electrospray ionization (ESI) probe. Proteomics analysis used a nanoLC coupled to a tribrid Orbitrap Lumos (Thermo Fisher Scientific, Bremen). Methods were performed as described previously [27 (link)–29 (link)]. Detailed mass spectrometry conditions and raw data alignment, filtering, and annotation parameters are described in (Additional file 1). For the assessment of repeatability and instrument stability over time, a QC strategy was applied. A quality control (QC) sample was prepared by pooling the same aliquot (10 µL) from each sample. Samples were injected in randomized order, and blank samples were injected regularly and used to assess carryover and exclude background signals.

Mass spectrometry data was acquired using an Orbitrap Lumos for all experiments apart from 6 fractions from the pSILAC_18 experiment, where an Orbitrap Fusion mass spectrometer was used instead (Thermo Fisher Scientific, San Jose, CA). In both cases, an Ultimate 3000 RSLC nano UHPLC equipped with a 300 μm ID x 5 mm Acclaim PepMap μ-Precolumn (Thermo Fisher Scientific) and a 75 μm ID x 50 cm 2.1 μm particle Acclaim PepMap RSLC analytical column was used.