Dionex ultimate 3000 rslc system

Pooled peptide fractions and individual plasma samples were re-suspended in 25 µl 2% acetonitrile/0.2% formic acid and spiked with iRT peptide standards (Biognosys, Zurich, Switzerland) as per manufacturer instructions. Analysis was performed on a Dionex Ultimate 3000 RSLC system (Thermo Fisher Scientific, Massachusetts, USA) coupled to a SCIEX 6600 TripleTOF mass spectrometer. Injected peptides (500 ng) were de-salted online using an Acclaim PepMap C18 trap column (75 μm × 2 cm) for 5.5 min at 5 μl/min using 2% acetonitrile/0.2% formic acid. Trapped peptides were separated on a Waters NanoEase™ C18 column (75 μm × 25 cm, 1.7 µm particle size). Peptides were eluted using a flow-rate of 300 nl/min and a gradient: 2–35% B over 45 min (A: 0.1% formic acid; B: 80% acetonitrile/0.1% formic acid). In DDA mode (pooled peptide fractions), precursor (MS) scans were acquired from m/z 400–1500 (2 + to 5 + charge states) using an accumulation time of 250 ms followed by 80 fragment ion (MS/MS) scans, acquired from m/z 100–1800 with 25 ms accumulation time each. For SWATH mode (individual plasma samples), precursor scan covered the range m/z 400 to 900 followed by 60 variable-width windows that overlapped by 0.5 Da, with fragment ions acquired from m/z 100–1800 with 25 ms accumulation time per window. See Supplementary Table 1 for summary of methods.

Size exclusion chromatography was performed on a Dionex Ultimate 3000RSLC system (ThermoScientific, Sunnyvale USA) with RI detection. The columns were TOSOH TSKgel G3000PWXL-CP (7.8 x 300 mm, 7 μm) and TOSOH TSKgel G-oligoPW (7.8 x3 00 mm, 7 μm) coupled in series and operated isocratically at 1 mL/min with 0.1 M NaNO₃ as the mobile phase. Samples were dissolved in the mobile phase. The system was calibrated with DIN-pullulan standards with molecular masses of 6 kDa, 12 kDa, 22 kDa, 50 kDa and 110 kDa (PSS Polymer Standards Service, Mainz, Germany). Chromatography data were exported and treated by WinGPC Scientific v 6.20 software for estimation of average molecular weights, degree of polymerization, average molecular mass, and dispersity [21 (link)].

SEC-HPLC and cation-exchange chromatography (CEX-HPLC) were employed to analyse the thermostability of trivalent IL-5-HSA Nb. Briefly, the trivalent IL-5-HSA Nb was diluted to 1 mg/mL and incubated at 2–8 °C or 25 °C for 1 month or even under 3 freeze-and-thaw cycles. After incubation, the stability of Nb was measured by SEC-HPLC and CEX-HPLC analyses. CEX-HPLC was performed on a Dionex Ultimate 3000 RSLC system (Thermo Fisher Scientific) with a BioMab NP5 PK column (Agilent Technologies).

Liquid chromatographic separation was performed on a Dionex UltiMate 3000 RSLC‐System (Thermo Fisher Scientific), using a 2.6 µm C18 (100 × 2.1 mm) UHPLC column (Kinetex Phenomenex), coupled online to a Q Exactive HF‐X (Thermo Fisher Scientific, Dreieich, Germany) orbital trapping mass spectrometer. The binary gradient was modified from the literature.
³⁶ (link) The solvent systems used were as follows: solvent A (60:40 acetonitrile: water, 0.1% formic acid, 10 mM ammonium formate [Sigma Aldrich]) and solvent B (90:8:2 isopropanol: acetonitrile: water, 0.1% formic acid, 10 mM ammonium formate [Sigma Aldrich, Germany]). The elution was performed with a gradient over 32 min. Starting condition was 32% of solvent B for 1.5 min. Until 4 min, solvent B was increased to 45%. From 4 to 5 min, solvent B was increased to 52%, from 5 to 8 min to 58%, from 8 to 11 min to 66%, from 11 to 14 min to 70%, from 14 to 18 min to 75% and from 18 to 21 min to 97%. From 21 to 25 min, the gradient was held constant. By the 26th minute, solvent B was reduced to 32% again. Subsequently, the gradient was kept unchanged for 7 min. The flow rate was kept constant at 260 µl/min throughout the whole measurement.

Size exclusion chromatography was performed on a Dionex Ultimate 3000RSLC system (ThermoScientific, Sunnivale USA) with RI detection. The columns were a TOSOH TSKgel G3000PWXL-CP (7.8 x 300 mm, 7 μm) and a TOSOH TSKgel G-oligoPW (7.8 x3 00 mm, 7 μm) coupled in series and where operated isocratically at 1 mL/min with 0.1 M NaNO₃ as the mobile phase. Samples were dissolved in the mobile phase. The system was calibrated with pullulan standards with molecular masses of 6 kDa, 12 kDa, 22 kDa, 50 kDa and 110 kDa (PSS Polymer Standards Service, Mainz, Germany). Chromatography data were exported and processed by WinGPC Scientific v 6.20 software for calculation of average molecular weight (MW) using linear calibration.

A Dionex Ultimate 3000 RSLC system (Thermofisher) combined with an Accucore aQ C₁₈ column (150 mm × 2.1 mm, 2.6 μm, Thermo fisher) was used in the sample separation process, and the separation speed was set at 0.3 μL/min for all the gradients. 1 μL of injection volume and a constant temperature of (25 ± 1) °C were adopted in the column. The eluents were A, water with 0.1% formic acid (v/v) and B, methanol, and the gradient program was as follows: 0–6 min, 8–40% B; 6–8 min, 40–100% B; 8–10 min, 100% B; 10–11 min, 100–8% B; 11–13 min, 8% B.