Nanoelectrospray ion source

Tryptic peptide fractions and lysates were reconstituted in 20 μL 3% acetonitrile/0.1% FA/doubly distilled water, and 1 ug per sample was loaded onto an EASY-nano-HPLC system (Proxeon) equipped with a RP-HPLC column (75 μm × 10.5 cm) packed in-house with 10 cm stationary phase (Magic C18 AQ 1.9 μm, 200 Å, Michrom BioResources). The HPLC was coupled to a QExactive plus MS (Thermo Scientific) equipped with a nano-electrospray ion source (Thermo Scientific). Peptides were loaded onto the column with buffer A (0.1% FA) and were eluted with 300 nL min⁻¹ buffer B (99.9% ACN, 0.1% FA). Subsequently, the column was washed with 98% buffer B. The tryptic peptide fractions were eluted with a 100 min gradient of 5–20% B followed by a 20 min gradient from 20–28% B, and a final 4-min step from 28–50% B. The column was washed with 98% buffer B. The MS was operated in data-dependent manner, with an automatic switch between MS to MS/MS scans. High-resolution MS scans were acquired (70,000, target value 10⁶) within 300–1700 m/z. The 15 most intense precursor ions were fragmented using higher-energy collisional dissociation (HCD) to acquire MS/MS scans (minimum signal threshold 420, target value 5 × 10⁴, isolation width 1.5 m/z). Unassigned and singly charged ions were excluded from HCD, and dynamic exclusion was set to 30 s.

All samples were analysed with the Easy-nLC system (Thermo Fisher Scientific), connected online to a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). Tryptic peptides were loaded in a fused silica column (75  µm inner diameter) packed with C18 resin (3-µm beads, Reprosil, Dr. Maisch), with solvent A (0.5% acetic acid). They were then eluted with a 120-minute gradient of solvent B (80% acetonitrile, 0.5% acetic acid) with a constant flow of 250 nL/min. The Q exactive was operated in positive mode with a capillary temperature of 250 °C, using the data-dependent acquisition method, which switches from full MS scans to MS/MS scans for the 12 most intense ions. Fragmentation was achieved by higher-energy collisional dissociation with a normalized collisional energy of 25. Full MS ranged from 300 to 1,750 m/z at a resolution of 70,000, an automatic gain control of 1e6, and a maximum injection time of 120 ms, whereas MS/MS events were scanned at a resolution of 35,000, an automatic gain control of 1e5, maximum injection time of 124 ms, isolation windows of 2 m/z, and an exclusion window of 45 seconds.

The dried immunoprecipitated peptides were resuspend in Buffer A (0.2% Formic Acid, 2% ACN, nanoLC grade 97.8% H₂O) and subjected to proteomic analysis using an EASY II nano-UPLC (Thermo Fisher Scientific) connected on-line to an Orbitrap Elite hybrid mass spectrometer with a nanoelectrospray ion source (Thermo Scientific) using settings similar to those previously described (Porras-Yakushi et al., 2015 (link)). Peptides were separated using a 15 cm silica analytical column with a 75 μm inner diameter packed in-house with reversed phase ReproSil-Pur C_18AQ 3 μm resin (Dr Maisch GmbH, Amerbuch-Entringen, Germany). The flow rate was set to 350 nl/min, using a linear gradient of 2%-32% B (0.2% Formic Acid, 80% ACN, 19.8% nanoLC grade H₂O). Mass spectrometry detectable samples were analyzed on a 159 min gradient, while basic reversed phase immunoprecipitated samples were analyzed on a 90 min gradient. The mass spectrometer was set to collect data in a data-dependent mode, switching automatically between full-scan MS and tandem MS acquisition. All samples were analyzed by ETD and decision tree fragmentation. For ETD fragmentation, the fifteen most intense precursor ions were selected, while the 20 most intense ions were selected for fragmentation using the decision tree method. Data acquisition was managed using Xcalibur 2.0.7 and Tune 2.4 software (Thermo Fisher Scientific).