1260 infinity lc system

Samples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes with a resolution of R(m/z = 200) = 280,000 for MS and R(m/z = 200) = 17,500 for MSMS experiments, in a single acquisition. MSMS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments (Surma et al., 2015 (link)). Both MS and MSMS data were combined to monitor CE, DAG, and TAG ions as ammonium adducts; PC, PC O-, as acetate adducts; and CL, PA, PE, PE O-, PG, PI, and PS as deprotonated anions. MS only was used to monitor LPA, LPE, LPE O-, LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts. Additional Cer and SM analyses (University of Potsdam) were carried out with a 1260 Infinity LC system coupled to a QTOF 6530 mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+). The precursor ions of Cer or SM species (differing in their fatty acid chain lengths) were cleaved into the fragment ions m/z 264.270 or m/z 184.074, respectively (Kachler et al., 2017 (link)).

AGP and water-soluble AX [average DP 131 (obtained using HP-SEC-MALLS using OHpak SB 802.5 HQ column on an Agilent 1260 infinity LC system)] were prepared from white flour from the wheat cultivar Yumai 34 using the method from Loosveld et al. [3 (link)] Fructo-oligosaccharides (FOS) from chicory (F8052 Sigma) (average DP 2–8) was used as a standard.

Approximately 300 μg of OMV from A. baumannii strain 19606 was diluted into 400 μL of PBS and applied to a PBS-equilibrated Superose 6 Increase 10/300 GL column (Agilent, Santa Clara, CA) connected to a 1260 Infinity LC System (Agilent). The absorbance at 210 nm was monitored. Fractions were collected every 0.5 mL starting at 5 mL. Fraction collection volumes were corrected for the void volume between the UV detector and the elution fraction collector of 1.692 mL. Fractions were directly coated (neat) in wells of microtiter plates for indirect ELISA (see below).

High-resolution mass spectra (HRMS) were collected on an Agilent 6530 Accurate-Mass Quadrupole Time-of-flight (QTOF) LC-MS instrument (Agilent Technologies) with an electrospray ionization (ESI) interface. Ten to 15 μl of each crude extract was injected onto a reversed-phase C₁₈ column [Phenomenex Luna 5-μm C18(2) column [4.6 × 150 mm; 100 Å]) on a 1260 Infinity LC-System (Agilent Technologies) using a flow rate of 0.75 ml/min to separate the analytes. MS analysis was performed by ESI in positive ionization mode (50 eV). A gradient from 80% solvent A (water with 0.1% formic acid) to 97% solvent B (acetonitrile [ACN] with 0.06% formic acid) was applied.

All solvents and chemicals were obtained from commercial sources and used without further purification. CAIX targeting agent 1 was synthesised in the laboratory as previously described [29 (link)]. IRDye 800 N-hydroxysuccinimide ester (NHS) (Biotium Inc., LI-COR, Fremont, CA) and agent 1 were conjugated following a previously reported method [30 (link)]. Briefly, agent 1, IRDye 800 NHS, and trimethylamine (in a 1:1:6 M ratio) were mixed in dimethylformamide and stirred at room temperature for 2 h. After the solvent was removed under vacuum, the product was purified by high-performance liquid chromatography. CAIX-800 was purified using an Inertsil C18 Luna 46 × 150-mm column on a 1260 Infinity LC system (Agilent, Santa Clara, CA). Mass spectroscopy was used to characterise the conjugates of the probe. The CAIX-FITC used for the in vitro cell binding assay was prepared and tested in the same way as CAIX-800.