Ltq orbitrap velos pro instrument

Between 2 and 10% of the collected fractions were analysed by LC–MS/MS using a nanoAcquity UPLC system (Waters Corporation, Manchester, UK) connected online to an LTQ-Orbitrap Velos Pro instrument (Thermo). Peptides were separated on a BEH300 C18 (75 μm × 250 mm, 1.7 μm) nanoAcquity UPLC column (Waters) using a stepwise 60 min gradient between 3 and 85% (v/v) ACN in 0.1% (v/v) FA. Data acquisition was performed using a TOP-20 strategy where survey MS scans (m/z range 375–1,600) were acquired in the Orbitrap (R=30,000) and up to 20 of the most abundant ions per full scan were fragmented by collision-induced dissociation (normalized collision energy=40, activation Q=0.250) and analysed in the LTQ Orbitrap. To focus the acquisition on larger cross-linked peptides, charge states 1, 2 and unknown were rejected. Dynamic exclusion was enabled with repeat count=1, exclusion duration=60 s, list size=500 and mass window ±15 p.p.m. Ion target values were 1,000,000 (or 500 ms maximum fill time) for full scans and 10,000 (or 50 ms maximum fill time) for MS/MS scans. All the samples were analysed in at least technical duplicates.

The 18 fractions were analyzed on a nanoAcquity UPLC system (Waters) directly connected to an LTQ Orbitrap Velos Pro instrument (Thermo Scientific) via a Proxeon nanospray source. The peptides were first trapped on a nanoAcquity Symmetry C18 5 µm 180 µm×20 mm (Waters) trapping column and further separated on a nanoAcquity BEH C18 1.7 µm 75 µm×200 mm (Waters) analytical column. The mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile (Biosolve). The applied three-step gradient was 120-min long, ranging from 3% to 40% acetonitrile. The flow rate was 300 nl/min. The eluent was directly introduced into the mass spectrometer through a Pico-Tip emitter 360-µm outer diameter×20-µm inner diameter, 10-µm tip (New Objective). The capillary temperature was set to 300°C, and the applied spray voltage was 2.2 kV. The mass range of the full scan MS spectra was 300–1700 m/z. MS1 spectra were recorded in profile mode in the Orbitrap. The resolution was set to 30,000. Lock mass correction was used for internal calibration using a background ion at m/z 445.12003. The 15 most abundant parent ions were subjected to fragmentation by using collision-induced dissociation. The normalized collision energy was set to 40. Charge state screening was enabled with only multiply-charged ions being selected for fragmentation.