Flexion nano spray apparatus

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-ultra performance liquid chromatography (Waters). The mobile phase was: (a) H₂O + 0.1% formic acid and (b) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; Waters). The peptides were then separated using a HSS T3 nano-column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; Waters). Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 35% B in 150 min, 35% to 90%B in 5 min, maintained at 95% for 5 min and then returned to initial conditions.
The nanoUPLC was coupled online through a nanoESI emitter (10 μm tip; New Objective, Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) using a FlexIon nano spray apparatus (Thermo Scientific).
Data were acquired in data dependent acquisition (DDA) mode, using a Top20 method. MS1 resolution was set to 70,000 (at 400 m/z), automatic gain control (AGC) to 3e⁶ and maximum injection time was set to 20 msec. MS1 isolation window was set to 1.6 mass units. MS2 resolution was set to 17,500, AGC to 1e⁶ and maximum injection time of 60 ms.

For the quantification of the trypsinized peptides of inflow and outflow segments of three dogfish specimens we used an ESI-Orbitrap-Quadrupole system. The Quadrupole-Orbitrap (Q Exactive Plus Thermo Scientific, Waltham, MA, USA) was coupled online through a nanoESI emitter (10 μm tip; New Objective; Woburn, MA, USA) using a FlexIon nanospray apparatus (Thermo Scientific, Waltham, MA, USA). Data were acquired using XCalibur v3.0 in data-dependent acquisition mode, using a Top20 method. Survey mass range was 300−1750 m/z. The quadrupole isolation window was set to 1.5 m/z; the AGC target was set to 1 × 106, a maximum injection time of 120 ms, and a resolution of 70000. In MS2 scans, target AGC was set to 5 × 105 with a maximum injection time of 60 ms. Protein quantification was achieved by the Label Free Quantification method, using the results from three different samples.