6230b lc tofms

The pH of the samples was set to 8.0 with the addition of 100 mM Tris. HCl and digestion with PNGase (Roche, Basel, Switzerland) were followed overnight at 37 °C for deglycosylation. Reduction was performed with 1.5 mM dithiothreitol (DTT) at 37 °C for 45 min, and samples were then analyzed immediately. Two µg of the samples were directly injected into an LC–ESI–MS system (LC: 1290 Infinity II UPLC, Agilent Technologies, Santa Clara, CA, USA). A gradient from 15 to 80% acetonitrile in 0.1% formic acid (using a BioResolve column (2.1 × 5 mm), Waters, Milford, MA, USA) at a flow rate of 400 μL/min was applied (9 min gradient time). Detection was performed with a Q-TOF instrument (6230B LC-TOFMS, Agilent Technologies) equipped with the Jetstream ESI source in positive ion, MS mode (range: 100–3200 Da). Instrument calibration was performed using an ESI calibration mixture (Agilent Technologies). Data were processed using MassHunter BioConfirm B.08.00 (Agilent Technologies), and the spectrum was deconvoluted with the MaxEnt algorithm.

The pH of the samples was set to 8.0 with the addition of 100 mM Tris. HCl and digestion with PNGase (Roche, Basel, Switzerland) were followed overnight at 37 °C for deglycosylation. Reduction was performed with 1.5 mM dithiothreitol (DTT) at 37 °C for 45 min, and samples were then analyzed immediately. Two µg of the samples were directly injected into an LC–ESI–MS system (LC: 1290 Infinity II UPLC, Agilent Technologies, Santa Clara, CA, USA). A gradient from 15 to 80% acetonitrile in 0.1% formic acid (using a BioResolve column (2.1 × 5 mm), Waters, Milford, MA, USA) at a flow rate of 400 μL/min was applied (9 min gradient time). Detection was performed with a Q-TOF instrument (6230B LC-TOFMS, Agilent Technologies) equipped with the Jetstream ESI source in positive ion, MS mode (range: 100–3200 Da). Instrument calibration was performed using an ESI calibration mixture (Agilent Technologies). Data were processed using MassHunter BioConfirm B.08.00 (Agilent Technologies), and the spectrum was deconvoluted with the MaxEnt algorithm.