Acquity 1 class uplc instrument

UHPLC/MS metabolomic analysis of placenta samples was performed with an Acquity I-class UPLC instrument and a Vion IMS QTOF mass spectrometer (Waters, Milford, MA, USA) using an Acquity UPLC HSS T3 column (150 × 2.1 mm, 1.8 µm, Waters) with a flow rate of 0.3 mL/min, an injection volume of 3 µL, an autosampler temperature of 8 °C, a column temperature of 30 °C and the following mobile phase gradient: 0 min - 100 % A, 2 min - 100 % A, 8 min - 100 % B, 10 min - 100 % B, 11 min - 100 % A, 20 min - 100 % A, where A was 0.1 % formic acid in water and B was 0.1 % formic acid in methanol. Electrospray ionization (ESI) full-scan mass spectra were acquired in positive-ion mode with the following tuning parameters: mass range 50-1000, soft transition mode, scan time 0.2 s, capillary voltage 0.5 kV, cone voltage 10 V, source offset 50 V, source temperature 130 °C, desolvation temperature 600 °C, cone gas flow 50 L/h, and desolvation gas flow 800 L/h. Leucine enkephaline was used as the lock mass for all experiments. Method validation based on selectivity, accuracy, precision, calibration curves, detection and quantification limits, matrix effects, extraction efficiency, and carryover (Supplementary Table 3) was performed as described previously (Lísa et al., 2017[36 (link)]).

The HPLC–MS measurements were performed on a Waters Acquity I-class UPLC instrument (Milford, MA, USA) coupled to a Waters Select Series Cyclic Ion Mobility (Milford, MA, USA) mass spectrometer. For the chromatographic separation of CS and HS disaccharides, a self-packed GlycanPac AXH-1 capillary column (250 µm i.d.) was used with the ammonium formate salt gradient methods published before [17 (link),18 (link)]. In the low-flow ESI ion source, the capillary voltage was set to 1.9 kV, while the cone voltage was 20 eV, and the temperature was 120 °C. The HS disaccharides were measured in MS1 mode, with the trap collision energy being 6 eV, and the transfer being 3eV. The CS was measured in MS1 and MS/MS modes, where the monosulfated isomer pairs were fragmented with 32 eV in the transfer to determine sulfation positions. Finally, the extracted ion chromatograms were integrated with the TargetLynx add-in of MassLynx software v4.2, Waters Corporation (Milford, MA, USA). The detailed integration method parameters are summarized in Table S2. Chromatogram examples are shown in Appendix A: Figure A1 (representative extracted ion chromatograms of CS disaccharides) and Figure A2 (representative extracted ion chromatograms of HS disaccharides).

Size-exclusion ultra-high-performance liquid chromatography (SE-UHPLC) analysis of rEqu c 1 wt and Triple variants were performed using Acquity BEH125 SEC column with dimensions of 4.6 × 150 mm, a pore size of 125 Å and a particle size of 1.7 µm (Waters) coupled with Acquity I-Class UPLC instrument (Waters) and controlled by Empower 3 software (Waters). The column was equilibrated to running conditions with PBS (12 mM Na₂HPO₄, 3 mM NaH₂PO₄, 150 mM NaCl, pH 7.3) as a mobile phase at a flow rate 0.3 ml/min until the baseline was stable. Samples of 2 µl of a 40 µM protein solution were injected. The chromatographic separation was carried out under isocratic flow with overall run time of 12 minutes and detection at 214 nm wavelength. The BEH125 SEC Protein Standard Mix (Waters) was used as a gel filtration standard.