Masshunter qualitative analysis b 06

The released N-glycans were quantitatively profiled on Chip-QQQ-MS using MRM method in positive mode. The dry-gas (N₂) temperature and flow rate were 225 °C and 11 l min⁻¹, respectively. The RF voltage amplitude of the high-pressure and low-pressure ion funnels were 150 and 200 V, respectively. The dwell time was set as 10–50 ms. All of the raw data were processed using Agilent MassHunter Qualitative Analysis B.06.00 and Agilent MassHunter Quantitative Analysis B.06.00 software. The optimized method was validated for linearity, sensitivity, recovery and repeatability. The abundance of each N-glycan composition was relatively quantified based on the peak areas of their MRM chromatograms and expressed as a percentage of summed peak areas for total N-glycans within each IgG samples.

The acquired chromatograms representing plasma metabolic fingerprints were extracted with the use of MFE algorithm provided by MassHunter Qualitative Analysis B.06.00 software (Agilent Technologies, Waldbronn, Germany). The parameters applied for data extraction were similar to the previously described (Ciborowski et al., 2014 (link)). The background noise threshold was set to 200 counts and the following adducts were included: +H, +Na, +K. Neutral water loss was also taken into account. After data extraction, each potential compound present in all plasma samples was described by the monoisotopic mass, retention time, and abundance.
Alignment of the chromatography data was performed with Mass Profiler Professional B.02.01 software (Agilent Technologies, Waldbronn, Germany) using 1% and 5 ppm for retention time and mass correction, respectively.
The aligned dataset was filtered based on the quality assurance (QA) criteria (Dunn et al., 2011 (link)) which included the presence of variables in at least 50% of QCs and the coefficient of variation (CV) value (< 20%) in QCs. A second filtering required the presence of the variable in 80% of the samples in at least one of the compared groups (i.e., in 80% of the samples in the responsive or non-responsive group, as well as in the PH patients or healthy individuals). These datasets were subsequently used for statistical analyses.

MS data were
collected using an Agilent 6540 QTOF mass spectrometer (Agilent Technologies)
equipped with a JetStream electrospray ion (ESI) source. Data acquisition
software was MassHunter Qualitative Analysis B.06.00 (Agilent Technologies).
The optimized operating parameters in negative ion mode were as follows:
nebulizing gas (N₂) flow rate at 7 L/min, nebulizing gas
temperature at 300 °C, JetStream gas flow at 7 L/min, sheath
gas temperature at 350 °C, nebulizer pressure at 40 psi, capillary
voltage at 3000 V, skimmer at 65 V, Octopole RFV at 600 V, and fragmentor
voltage at 130 V. An MS/MS technique was applied to provide parallel
alternating scans for acquisition at low collision energy to obtain
precursor ion information or at a ramping of high collision energy
to acquire a full-scan accurate mass data of fragments and precursor
ions and to obtain neutral loss information. The collision energies
for auto MS/MS analysis were 20 and 35 V.