Lc 20 system

Lyophilized mucus was dissolved in 30% acetonitrile (AcN) in water and 0.1% formic acid (FA), and desalted using size exclusion chromatography (SEC; PD-10, GE Healthcare). The eluate was lyophilized and redissolved in 10% AcN, 0.1% FA, and subjected to RP-HPLC on a Phenomenex Jupiter column (5 µ C18 300 Å, 250 × 4.6 mm) using a Shimadzu LC20 system. The gradient ranged from 5 to 55% AcN, in 0.05% trifluoroacetic acid (TFA).

Plasma concentrations of Trp, 5-HT, KYN and KYNA were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved on an Atlantis dC18 reverse phase column (C18, 3 μm, 20 × 4.6 mm (int. diam.)) and elution with (A) HCO₂H 0.1% (for positive mode on MS/MS), for 1 min and from A to (B) CH₃CN: A (40:60, v/v) in 2 min and for 2 min in B on a Shimadzu LC20 system with a quaternary pump. MS/MS was performed on a Shimadzu LC-MS/MS 9030 system (Shimadzu, Japan). Internal standards for Trp, 5-HT, KYN and KYNA were purchased from Sigma (Sigma-Aldrich, China).

The experimental C_p/C₀ values of NKT were determined by RP-HPLC/MS. The column used was a C18 Prosphere (250 × 21 mm, 5 µm diameter beads) provided by Alltech (Carquefou, France) connected to the Shimadzu model LC20 system. For the analysis, the column was kept at 35 °C. A total of 10 µL of sample was injected. For the elution, gradient of solvent A (water/acetonitrile in 94.9/5 proportion (v/v) with 0.1% TFA (v/v)) and solvent B (water/acetonitrile in 4.9/95 proportion (v/v) with 0.1% TFA (v/v)) was used. The starting condition was 100% A. A first slope was applied to reach 28% B/72% A in 50 min. Then, a second slope was applied to reach 48% B/52% A in 20 min. The column was finally washed with 100% B for 10 min and re-equilibrated in 100% A for 15 min. The flowrate was 0.2 mL·min⁻¹. The eluent was analysed on-line by electrospray ionization mass spectrometry (ESI-MS) (Shimadzu Corporation, Kyoto, Japan) in positive mode. The following operating parameters were used in TIC mode: mass scan = 50–2000 m/z, ion spray tension = 4.5 kV, heat block temperature = 200 °C, drying gas flow = 15 L/min, scan speed = 2143 mass units/s. Selected Ion Monitoring (SIM) mode was also used to monitor the targeted peptides. The integration values of NKT MS signal were integrated to assess the C/C₀ values in the UF samples.

Ultra-fast liquid chromatography (UFLC) analysis of flavonoids was conducted on an LC–20 system (Shimadzu) linked to an SPD–M20A instrument. A 10 µL aliquot of the sample solution was injected into a reversed phase 250 mm×4.6 mm, ID 5 µm ZORBAX SB-C18 column (Agilent Technologies, USA). The mobile phase was composed of (A) 0.1% aqueous formic acid and (B) methanol. Gradient elution was performed as follows: 0–20 min, 37%–50% B; 20–35 min, 50%–80% B; and 35–40 min, 80%–100% B. The column thermostat was set to 25°C and the flow rate was 0.7 mL/min, similar to the conditions specified in our previous report [17] . The detection wavelength was set to 283 nm for flavanones, 330 nm for flavones, and 367 nm for flavonols.
All flavonoids were quantified with external standards by UFLC analysis. The concentrations of flavonoids were expressed as micrograms per milliliter solution volume (µg/mL). Fourteen different standard stock solutions with varying concentrations were prepared. The linear regression equation obtained within the range of 2.0 µg/mL to 20 µg/mL showed an R²≥0.996 for all of the measured flavonoids. The repeatability of intraday analysis showed a relative standard deviation (RSD) of≤3% (n = 3). Among the flavonoids, hesperidin showed the maximum limit of detection at 0.04 µg/mL, and narirutin showed the minimum limit of quantitation at 0.05 µg/mL.

The GC–FID analyses were carried out with capillary GC using an Agilent 6890N GC system (Agilent, Santa Clara, CA, USA), and the GC/MS analyses were performed on an Agilent 5975 GC–MSD system (Agilent, Santa Clara, CA, USA). An HP-Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness, Agilent, Wilmington, DE, USA) was used for the analyses. The HPLC chromatographic separations were carried out using Shimadzu LC 20 System (Shimadzu, Tokyo, Japan). The mass spectra were recorded with AB Sciex 3200 Q TRAP mass spectrometer (AB Sciex, Toronto, Canada). GL Science Inertsil ODS 250 × 4.6 mm, 5 μm i.d. particle size, analytical column (GL Sciences, Tokyo, Japan) was used for the HPLC analyses. The turbidity of the standardized microbial sample solutions was measured using McFarland densitometer (Biosan McFarland Densitometer, Model Den-1B, Riga, Latvia). Antioxidant activity absorbances were recorded with a Biotek microplate reader (BioTek, Winooski, Vermont, USA). Chlorogenic acid, luteolin 7-glucoside, gallic acid, butylated hydroxytoluene (BHT), and L-ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

We dissolved angiotensin II to a concentration of 2 mg/mL in water (H₂¹⁶O) or in hydrogen heavy oxide (H₂¹⁸O). The pH of the solution was 6.29. We performed all the irradiation experiments in a 0.1 cm quartz cuvette light path. We irradiated the angiotensin II solutions for 0 and 60 min with UVC emitted from the PDUVL at room temperature. After irradiation, we submitted a five-fold diluted solution (5 μL) to an LC20 system (Shimadzu, Kyoto, Japan) with an ACQUITY UPLC Peptide BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 150 mm) at a column temperature of 50 °C. We carried out the reversed-phase liquid chromatography (LC) separation as described above, with a slight modification of the gradient of the mobile phase B (from 2% to 80%, 13 min).
The eluates were introduced directly into an ESI-MS system consisting of a Q-TOF mass spectrometer, maXis II™ ETD (Bruker Daltonics, Billerica, MA). We calibrated the mass values by using ESI-L Low-Concentration Tuning Mix (Agilent Technologies, Palo Alto, CA). All the measurements were performed in the positive-ion mode. We analyzed the peptide fragments using data-analysis software (Bruker Daltonics).

To assess the activity of LtgA, PG (200 µg) was incubated in the presence of LtgA, or equimolar amounts of LtgA and Ape1, in 12.5 mM sodium phosphate buffer pH 5.6. Neisseria PG was purified as previously described (Wheeler et al., 2014 (link)). The reaction mix was initiated by the addition of enzymes and incubated at 37°C for 5 min. Control reactions lacking PG or enzyme/inhibitor were also included. The final reaction volume was 200 µL. Reactions were performed in triplicates. The reaction was stopped by incubating the samples in a heat block at 100°C for 5 min. The soluble 1,6-anhydro-muropeptides was collected using centrifugation at 16,000 g for 10 min at room temperature. The supernatant was collected and analyzed by reversed-phase HPLC using a Shimadzu LC-20 system with a Hypersil GOLD aQ column (5 μm particle size, 250 × 4.6 mm, flow rate 0.5 mL/mL at 52°C; Thermo Fisher Scientific (Waltham, MA, USA). The mobile phase gradient was 50 mM sodium phosphate pH 4.3 to 75 mM sodium phosphate pH 4.9 with 15% Methanol over 135 min.