Easy spray pepmap rslc

All peptide separations were carried out using an Ultimate 3000 nano system (Dionex, Thermo Fisher Scientific). For each analysis the sample was loaded onto a trap column (Acclaim PepMap 100, 2 cm × 75 μm inner diameter, C18, 3 μm, 100 Å) at 5 μL/min with an aqueous solution containing 0.1% (v/v) TFA and 2% (v/v) acetonitrile. After 3 min, the trap column was set in-line with an analytical column (Easy-Spray PepMap® RSLC 15 cm × 75 μm inner diameter, C18, 2 μm, 100 Å (Dionex). Peptide elution was performed by applying a mixture of solvents A and B. Solvent A was HPLC grade water with 0.1% (v/v) formic acid, and solvent B was HPLC grade acetonitrile 80% (v/v) with 0.1% (v/v) formic acid. Separations were performed by applying a linear gradient of 3.8% to 50% solvent B over 30 min at 300 nL/min followed by a washing step (5 min at 99% solvent B) and an equilibration step (15 min at 3.8% solvent B).

Peptides were analysed by LC-MS/MS using an Ultimate 3000 nano system (Dionex/Thermo Fisher Scientific, Hemel Hempstead, UK) coupled to a Q-Exactive-HF mass spectrometer (Thermo Fisher Scientific, Hemel Hempstead, UK) to acquire the masses of peptides derived from proteins extracted from the BALF samples. Peptides were loaded onto a trap column (50 cm) (Acclaim Pepmap 100) at 12 µl/min over 7 min with an aqueous solution containing 0.1% (v/34v) TFA and 2% (v/v) acetonitrile. The trap column was set in-line with an analytical column (Easy-Spray Pepmap® RSLC) (Dionex). Peptides were eluted by applying a linear gradient of 3.8–50% solvent B (acetonitrile 80% (v/v) with 0.1% (v/v) formic acid FA over 35 min at 300 nl/min. Solvent A was HPLC grade water with 0.1% (v/v) formic acid. The mass spectrometer was operated in data dependent positive (ESI+) mode and full scan MS spectra (350–2000 m/z) were acquired in the Orbitrap. The 16 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the octopole collision cell by high energy collisional dissociation (HCD) and detected in the Orbitrap.

LC-MS/MS analysis was conducted on a Dionex 3000 coupled in line to a Q-Exactive-HF mass spectrometer. Digests were loaded onto a trap column (Acclaim PepMap 100, 2 cm × 75 µm inner diameter, C18, 3 microM, 100 ˚A) at 5 µL per min in 0.1%(v/v) TFA and 2%(v/v) acetonitrile. After 3 min, the trap column was set in line with an analytical column (Easy-Spray PepMap® RSLC 15 × 50 cm inner diameter, C18, 2 microlM, 100 ˚A) (Dionex). Peptides were loaded in 0.1%(v/v) formic acid and eluted with a linear gradient of 3.8–50% buffer B (HPLC grade acetonitrile 80%(v/v) with 0.1%(v/v) formic acid) over 95 min at 300 nl per min, followed by a washing step (5 min at 99% solvent B) and an equilibration step (25 min at 3.8% solvent). All peptide separations were carried out using an Ultimate 3000 nano system (Dionex/Thermo Fisher Scientific). The Q-Exactive-HF was operated in data-dependent mode with survey scans aquired at a resolution of 60,000 at 200 m/z over a scan range of 350–2000 m/z. The top 16 most abundant ions with charge states +2 to +5 from the survey scan were selected for MS2 analysis at 60,000 m/z resolution with an isolation window of 0.7 m/z, with a (N)CE of 30%. The maximum injection times were 100 and 90 ms for MS1 and MS2, respectively, and AGC targets were 3e6 and 1e5, respectively. Dynamic exclusion (20 s) was enabled.