Synapt g2 si hdms system

Separation of the antibodies using size-exclusion chromatography was accomplished using a Waters Alliance 2695 (Waters, Milford, MA, USA) connected to a BioSuite High Resolution SEC Column (7.5 mm × 300 mm, 10 µm particle size, Waters, Milford, MA, USA). The separation was performed using an isocratic elution with PBS, pH 7.0, at a flow rate of 1 mL/min. The effluent was detected with a UV/Vis detector 2469 at 280 nm.
The intact masses of the antibodies were determined using reverse-phase chromatography separation. The separation for intact mass was accomplished using a Waters ACQUITY I class UPLC system (Waters, Milford, MA, USA) connected to a Thermo MabPac^TM RP column (2.1 mm × 50 mm, 4 µm particle size, Thermo Fisher Scientific). The separation was performed with eluent A, consisting of 0.1% formic acid in water, and eluent B, consisting of 0.1% formic acid in 100% acetonitrile, at a flow rate of 0.2 mL/min. The gradient was fixed for 2 min at 20% eluent B and linearly increased for 8 min from 20% to 50% eluent B. The effluent was analyzed with a Waters Synapt G2-Si HDMS system. A quantity of 50 μL (0.2 mg/mL) of antibodies was mixed with 1.5 μL (500,000 units/mL) of PNGaseF (New England BioLabs), then reacted overnight at 37 °C. Native and PNGaseF-treated antibodies were injected with 5 μL of the samples.

A Waters Synapt G2-Si HDMS
system equipped with
a prototype uMALDI source and provided with a Nd:YAG laser (Waters
Corporation, UK) was used for online recognition experiments. For
more detailed information about the uMALDI source, see Barré
et al.^{23 (link)} Data acquisition was performed
using MassLynx version 4.1 and HDImaging version 1.5 software (Waters
Corporation). For online recognition, our model was built using the
AMX Model Builder, which was loaded into AMX recognition software
that was coupled to the data acquisition file. All the measurements
were performed in the sensitivity mode with a scan rate of 1.0 s per
scan, trap collision energy (CE) of 4, and transfer CE of 2, 1000
Hz laser repetition rate, and mass range of m/z 300–1200 in the positive ion mode. The instrument
was calibrated with red phosphorus for the positive ion mode before
each measurement. The spatial resolution was 30 × 30 μm².