Trap column

Nanoscale LC separation of tryptic peptides was performed using a nanoACQUITY^TM system (Waters) equipped with a nanoEase^TM 5 mm × Bridge^TM BEH130 C18 300 mm × 50 mm precolumn; trap column 5 mm, 180 mm × 20 mm; and BEH130 C18 1.7 mm, 100 mm × 100 mm analytical reversed-phase column (Waters). The peptides were separated into 10 fractions and the gradient elution was performed as follows: 8.7, 11.4, 13.2, 14.7, 16, 17.4, 18.9, 20.7, 23.4, and 65% acetonitrile/0.1% (v/v) formic acid, with a flow rate of 2000 mL/min. The source was operated in positive ionization mode nano-ESI (+). GFP [Glu]¹-fibrinopeptide B human ([MC2H]²⁺ = 785.8426) (Millipore Sigma) was used for lock mass calibration of the apparatus, using a constant flow rate of 0.5 μL/min at a concentration of 200 fmol protein. MS analysis was performed on a Synapt G1 MS^TM (Waters) equipped with a NanoElectronSpray source and two mass analyzers: a quadrupole and a time-of-flight (TOF) operating in V-mode. The mass spectrometer was programmed in the data-dependent acquisition mode, in which a full scan in the m/z region of 50–2000 was used. Data were obtained using the instrument in the MS^E mode, which switched between the low energy (6 V) and elevated energy (40 V) acquisition modes every 0.4 s. Samples were analyzed using three replicates.

LC-MS/MS analysis of iTRAQ-labeled peptides was performed on a Synapt G2 mass spectrometer interfaced with a nanoacquity UPLC nanoflow liquid chromatography system (Waters, Miliford, MA, USA). The fractions were enriched on a trap column (180 µm × 2 cm, 5 µm, Waters, Miliford, MA, USA) at a flow rate of 8 µl/min for 5 min and then resolved on an analytical column (75 µm × 15 cm, 1.7 µm, Waters, Miliford, MA, USA) with the application of a voltage of 3Kv. The peptides were eluted using a linear gradient of 7–30% solvent B (90% acetonitrile in 0.1% formic acid). LC-MS/MS data were acquired using positive ion mode in a data-dependent manner from m/z 300 to 1600Da, targeting the three most abundant ions in the survey scan. MS data were acquired in the QTOF analyzer, and MS/MS spectra were acquired for 1.5 seconds. Collision-induced dissociation mode (CID) was used for MS/MS scans.