Q excative hf mass spectrometer

The protein secretome in conditioned media of treated cells was analysed by Electrospray ionization LC-MS. Samples from two independent experiments were run on an Ultimate 3000 RSLC system (Thermo Scientific, Sunnyvale, California, United States) connected online to a Q-Excative HF mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with EASY-spray nano-electrospray ion source source (Thermo Scientific). MS spectra (from m/z 375–1500) were acquired in the Orbitrap with resolution R = 60000 at m/z 200, automatic gain control (AGC) target of 3e6 and a maximum injection time (IT) of 110 ms. Complete methods are described in detail in supplementary methods.

Lyophilised Tryptic peptides (Centrivap with a Cold trap, Labconco, MO) were reconstituted in 1% aqueous formic acid (FA) / 2% acetonitrile (ACN), and 1 μg of each sample was automatically injected into an Ultimate 3000 RSLC system (Thermo Scientific, Sunnyvale, CA) connected online to a Q-Excative HF mass spectrometer (Thermo Scientific, Bremen, Germany). Peptides were pre-concentrated on a pre-column (Acclaim PepMap 100, 2cm x 75μm i.d. nanoViper column, packed with 3μm C18 beads), before separation of peptides on a 50 cm analytical column (Acclaim PepMap100 nanoViper column, 75 μm i.d. × 50 cm, packed with 3 μm C18 beads) at a flow rate of 250 nl/min. The peptides were separated utilising a biphasic gradient with 0.1% FA (solvent A) and 100% ACN (solvent B) for 120 min (5% B during trapping for 5 min followed by 5–7% B over 0.5 min, 7–22% B over 59.5 min, 22–35% B over 22 min, 35–90% over 5 min and then hold at 90% B for 10 min, then sloped to 5% B over 3 min and hold at 5% B for 15 min).