Dionex ultimate 3000 hplc

HSA fusion proteins were produced by transient transfection of HEK293E cells. Transfected cells were grown in serum-free 293Freestyle media (Gibco, #12338018). Secreted protein was purified from supernatant using CaptureSelect Human albumin affinity matrix (ThermoFisher, #191297005) by elution with 2 M MgCl₂, pH 7.4. Purified protein was concentrated and buffer exchanged into PBS by Vivaspin2 centrifugal concentrators (Sartorius). The bispecific LiTE protein was expressed and purified by Nickel-NTA followed by protein A affinity chromatography, as described elsewhere^{26 (link)}. For size exclusion chromatography the protein sample in 100 mM Tris-Hcl pH 7.4 was run using a Yarra™ 1.8 µm SEC-X150 column (Phenomenex) on a Dionex Ultimate 3000 HPLC (ThermoFisher Scientific) with a flowrate of 0.1 mL per minute. A protein standard ranging from 670 kDa to 0.244 kDa (Phenomenex) was run with the same settings and overlaid for comparison.

This step was performed as described previously (40 (link)), modified and extended to also allow quantification and fragmentation of glycerophosphocholines and glycerophosphoethanolamines. Internal standards were added to cell homogenates, and lipids were extracted via the Folch procedure (41 (link)). Dried lipid extracts were dissolved in HPLC starting condition, separated by reversed-phase HPLC on a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific), and quantified with a Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific). Baseline corrected data were integrated in MZmine 2 (42 (link)), quantified, and visualized as described previously (40 (link)) using custom-made scripts in R (https://www.R-project.org/). Molecular PE, PC, plasmanyl-PE and plasmenyl-PE species were identified by their retention time, monoisotopic mass-to-charge ratio, isotope pattern, and fragmentation behavior (SI Appendix, Fig. S5 and Materials and Methods), which was cross-validated with single lipid standards commercially available from Avanti Polar Lipids: C16-18:1 PC, C18(Plasm)-22:6 PC, C16-18:1 PE, C18(Plasm)-18:1 PC, C18(Plasm)-18:1 PE, and C18(Plasm)-20:4 PC.

MDA in the colon was extracted and measured by high-performance liquid chromatography-fluorescence detection (HPLC-FLD) as previously described with minor modifications [32 (link)]. Briefly, 50 mg of colon was homogenized in 0.5 mL ice-cold 0.15 mol/L KCl containing 0.01% BHT using a Fisher Scientific™ Bead Mill 4 homogenizer (Pittsburgh, PA, USA). Then, 50 µL homogenate was mixed with 400 µL of KCl, 40 µL of 0.2% BHT in ethanol and 200 µL of 1N NaOH, and then incubated at 60 °C for 30 min. Then, protein was precipitated by mixing with 2 mL of 5% TCA. After centrifugation at 1000× g for 10 min at 4 °C, 500 µL of supernatant was derivatized with 0.6% TBA at 95 °C for 30 min. Then, the sample was extracted with butanol, and injected onto a Dionex UltiMate 3000 HPLC equipped with an LPG-3400 quaternary pump, a WPS-3000 analytical autosampler, and an FLD-3100 fluorescence detector (Thermo Fisher Scientific, San Jose, CA, USA). Samples were resolved isocratically at 0.8 mL/min on a Discovery^® C18 column (250 × 4.6 mm, 5µm; Supelco, Bellefonte, PA, USA) using 40:60 methanol and 25 mM potassium phosphate buffer (pH 6.5). MDA was quantified against standards prepared in parallel from TMP and was normalized to colonic protein.

The LC setup was as described above, but a Dionex Ultimate 3000 HPLC (Thermo Scientific, Dreieich) and a 350 mm capillary C18 column were used.
Both mass spectrometers were operated in data-dependent acquisition mode with a survey scan resolution of 120 000 (for Orbitrap Fusion) or 60 000 (for Q-Exactive HF) at m/z 200, with an AGC target of 1 x 10e6. Up to 30 of the most intense precursor ions with charge state 2 or higher were sequentially isolated for HCD with normalized collision energy of 27% (Q Exactive HF) or 30% (Orbitrap Fusion), respectively. MS/MS scans were recorded in the Orbitrap for Q Exactive HF, and in the LTQ ion trap for the Orbitrap Fusion. Dynamic exclusion was set to 50 s.

The culture broth was centrifuged at 13,500 × g for 2 min, and the supernatant was analyzed for aromatic compound concentrations using a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific, MA, USA) equipped with a XBridge BEH C18 250 mm×4.6 mm column (particle size 5 μm) (Waters, MA, USA). L-phenylalanine, phenylacetate, 2-phenylethanol and phenylpyruvate samples were analyzed with 70% solvent A (water with 0.1% formic acid) and 30% solvent B (acetonitrile) at a flow rate of 0.5 mL/min with UV detector under 210 nm. L-phenylalanine was detected at 7.5 min, phenylpyruvate at 3.5 min, 2-PE at 13.3 min and phenylacetate at 5.3 min. All the peak areas of these aromatic compounds were integrated and used for quantification by fitting with standard curves. Phenylacetate was not detected in all the samples of strains, so that the results of phenylacetate were omitted.
The quantitation of glucose, ethanol, glycerol and acetate were determined by a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific, MA, USA) equipped with an Aminex HPX-87G column (Bio-Rad, CA, USA). The column was eluted with 5 mM H₂SO₄ at a flow rate of 1.0 mL/min at 50°C for 22 min. Glucose, ethanol, acetate and glycerol were detected by RI detector at 9.6 min, 17.9 min, 14 min and 12.9 min, respectively. For all samples, at least three biological replicates were analyzed.

Semi-untargeted qualitative and quantitative analyses were performed by an HPLC system (Dionex ultimate 3000 HPLC, ThermoFisher Scientific, Waltham, MA, USA) coupled via atmospheric-pressure chemical ionization (APCI) to a high-resolution tandem mass spectrometry instrument (HRMS²; LTQ Orbitrap, ThermoFisher Scientific, Waltham, MA, USA). Separation was carried out with Luna C18(2) 150 × 2 mm, 100 Å, 3 µm (Phenomenex, Torrance, CA, USA) using 0.1% (v/v) formic acid (solvent A) and 0.1% (v/v) acetonitrile (solvent B) as mobile phases. Chromatographic separation consisted of a solvent ramp from 5% to 100% solvent B for 30 min, followed by column reconditioning of 15 min. The flow rate was set to 0.2 mL min⁻¹ and the injection volume to 10 µL. Detection parameters were the following: negative ionization mode; capillary temperature: 250 °C; APCI vaporizer temperature: 450 °C; sheath gas: 35 Arb; auxiliary gas: 15 Arb; discharge needle: 5 kV; the acquisition was carried out in Dependent Scan mode with a mass range from 220 to 1000 m/z, normalized CE: 35V. Raw data obtained were analyzed with Thermo Xcalibur software (ThermoFisher Scientific, Waltham, MA, USA); molecules were identified using the MetFrag online tool [90 (link)]. Quantification of identified acidic triterpenes was performed with a calibration curve of moronic acid (TCI-Europe, Bruxelles, Belgium, EU).

For the determination of the exact mass, the saponin extract was injected in a high-resolution LC/MS system. The liquid chromatograph was Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved in the same conditions than low-resolution LC-MS (previous section). A split post-column of 0.4 mL/min was introduced directly on the mass spectrometer ion source. Mass spectrometry was performed using a micrOTOF-QII High-Resolution Time-of-Flight mass spectrometer (UHR-TOF) with qQ-TOF geometry (Bruker Daltonics, Bremen, Germany) equipped with an ESI. The instrument was operated in negative-ion mode using a scan range of m/z 50–1200. Mass spectra were acquired through broad-band Collision-Induced Dissociation bbCID mode, providing MS and MS/MS spectra, simultaneously. The instrument control was performed using Bruker Daltonics HyStar 3.2 (Bruker bioscience España, Sevilla).