Acclaim pepmap μ precolumn

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.

The OMV preparations from three independent biological replicates were separated in 10‐μl aliquots, each, by SDS–PAGE. Three zones were excised per lane and proteins were S‐alkylated with iodoacetamide and subsequently subjected to in‐gel digestion with modified trypsin (Promega, Mannheim, Germany). The peptide mixture was analyzed in the positive‐ion DDA mode (switching to MSMS mode for eluting peaks) using a Dionex Ultimate 3000 system (Thermo Scientific, Vienna, Austria) directly linked to a quadrupole time‐of‐flight mass spectrometer (MS) (Bruker maxis 4G ETD; Bruker) equipped with the captive spray source and nano‐booster set‐up. MS scans were recorded over a range of 150–2200 Da and the eight highest peaks were selected for fragmentation. Instrument calibration was performed using the ESI calibration mixture (Agilent, Santa Clara, CA). For separation of the peptides, a Thermo Acclaim PepMap300 RSLC C18 separation column (2‐μm particle size; 150 × 0.075 mm) was used with a Thermo Acclaim PepMap μ‐pre‐column. A gradient from 5% to 32% of solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1% formic acid in HQ‐water) within 60 min was applied at a flow rate of 0.3 μl min⁻¹, followed by a 10‐min gradient from 32% B to 70% B to promote elution of large peptides.