Glu1 fibrinopeptide b

Peptides were loaded on reversed-phase trap column PST C18, 100 Å, 5 μm, 180 μm × 20 mm (Waters Corporation, UK) with a flow rate of 15 μL/min using loading buffer of 0.1% formic acid and subsequently separated on HSS-T3 C18 1.8 μm, 75 μm × 250 mm analytical column (Waters Corporation, UK) in 90 min linear gradient (A: 0.1% formic acid, B: 100% CH₃CN, and 0.1% formic acid) at a flow rate of 300 nl per min.
The nano-LC was coupled with HDMS Synapt G2 mass spectrometer (Waters Corporation, UK). Data were acquired using Masslynx version 4.1 software (Waters Corporation, UK) in positive ion mode. LC-MS data were collected using data independent acquisition (DIA) mode MSE in combination with online ion mobility separation. The trap collision energy of mass spectrometer was ramped from 18 to 40 eV for high-energy scans in MSE mode. The trap and transfer collision energy for high-energy scans in HDMS mode was ramped from 4 to 5 eV and from 27 to 50 eV. For both analyses, the mass range was set to 50–2,000 Da with a scan time set to 0.9 seconds. A reference compound [Glu1]-fibrinopeptide B (Waters Corporation, UK) was infused continuously (500 fmol/μL at flow rate 500 nL per min) and scanned every 1 minute for online mass spectrometer calibration purpose. The samples were run in triplicate.

Ten microlitres (10 μL) of reconstituted released N-glycans were injected into an ACQUITY H-Class UPLC (Waters Corporation, United States) coupled to a Xevo G2S QTof mass spectrometer (Waters Corporation, United States). Samples were separated using an ACQUITY UPLC Glycan BEH amide column (130 Å, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation, United States) at 60°C and 400 μL/min, with a 40 min gradient from 25 to 49% of 50 mM Ammonium Formate (mobile phase A). 100% ACN was used as mobile phase B. RFMS-labelled glycans were excited at 265 nm and measured at 425 nm with an ACQUITY UPLC FLR detector (Waters Corporation, United States). The MS1 profile scans of m/z 400–2,000 were acquired using the Xevo G2S-QTof in positive mode with an acquisition rate of 1 Hz. The electrospray ionisation capillary voltage was set at 2.75 kV, cone voltage at 15 V, desolvation gas flow at 800 L/h, ion source temperature and desolvation temperature were kept at 120°C and 300°C, respectively. Glu1-fibrinopeptide B (Waters Corporation, United States) was used as the LockSpray compound for real-time mass correction. RapiFluor-MS Dextran Calibration ladder (Waters Corporation, United States) was also injected into LC-MS to calibrate the retention time of sample peaks. The retention times were normalised using the dextran calibration curve to Glucose Units (GU).