Agilent series 6560 lc ims qtofms

To quantify the degree of underglycosylation of plant produced IgG1 via mass spectrometry purified proteins were either measured intact or the fraction of unglycosylated heavy chain analyzed from trypsin-digested glycopeptides. For the intact measurement, 1 µg of protein was applied directly to a Waters BioResolve Column (2.1 x 5 mm). An acetonitrile gradient was used ranging from 15% to 80% (v/v) acetonitrile in 0.1% (v/v) formic acid. Gradient time was 15 minutes, flow rate was set to 400 µL/min at 80°C. Detection was performed on a Q-TOF instrument (Agilent Series 6560 LC-IMS-QTOFMS) equipped with the JetStream ESI source in positive mode. Data analysis was done using MassHunter BioConfirm B.08.00. Spectrum deconvolution was performed using MaxEnt. For quantification on the peptide level, glycopeptides were deglycosylated using Protein-N-glycosidase A (PNGase A, Europa Bioproducts Ltd) which leads to deamidation of the glycosylated asparagine residue present in the N-glycosylation site. The resulting mass shift of + 0.984 can be monitored via mass spectrometry. For normalization, a synthetic peptide EEQYNSTYREEQYDSTYR (JPT Peptide Technologies) was used as described (Castilho et al., 2018 (link)). Analysis of mass spectrometry data was done using Skyline Version 22.2. ANOVA with Tukey post-hoc tests were carried out using GraphPad Prism 9.2 software.

As previously described (Michlits et al., 2020 (link)), H₂O₂ induced conversion of coproheme to heme b was investigated by analyzing the same reaction mix in UV–Vis absorbance and by mass spectrometry. Typically, 1,000 µL enzyme solution in reaction buffer (RB: 50 mM phosphate buffer, pH 7) with around 15 μM apoenzyme and 10 μM coproheme was titrated with subequimolar amounts of hydrogen peroxide (4 mM H₂O₂ stock in RB) in a Cary 60 spectrophotometer (Agilent Technologies). About 10 µl samples from this reaction mix were drawn and directly taken to MS analysis after taking UV–Vis spectra. About 4 µl of the protein solution was directly injected into an LC-ESI-MS system (LC: Agilent 1290 Infinity II UPLC). A gradient from 15 to 80% acetonitrile in 0.1% formic acid (using a Waters BioResolve column (2.1 × 5 mm)) at a flow rate of 400 μl/min was applied (15 min gradient time). Detection was performed with a Q-TOF instrument (Agilent Series 6560 LC-IMS-QTOFMS) equipped with the Jetstream ESI source in positive ion, MS mode (range: 100–3,200 Da). Instrument calibration was performed using the ESI calibration mixture (Agilent Technologies). Data were processed using MassHunter BioConfirm B.08.00 (Agilent Technologies) and the spectrum was deconvoluted by MaxEnt.