Acquity uplc peptide beh c18 column

Fecal lipidomic profiling was performed at the Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences, as described previously (62 (link)). Briefly, collected mouse feces were homogenized in methanol, and lipids were obtained by single-phase extraction. Lipids were separated on an Acquity UPLC Peptide BEH C18 column (Waters). Oxidized fatty acids, including LA metabolites, were extracted using a Sep-Pak C18 cartridge (Waters) and analyzed by LC-quadrupole MS (QTRAP4500 or QTRAP5500; Sciex). Untargeted lipidomics was performed using an ACUITY UPLC system (Waters) coupled with QTOF-MS (TripleTOF 5600⁺ or TripleTOF 6600; Sciex) (93 (link)).

The peptide content of APE was measured by the Folin phenol reagent method [26 (link)]. The sugar content of APE was detected at 490 nm using the phenol–sulfuric acid method [27 (link)]. The pH of APE was measured using a pH meter (S400-B, METTLER TOLEDO, Columbus, OH, USA). In addition, the maximum absorption wavelength of APE was measured using a UV spectrophotometer. Finally, the peptide sequences of APE were analyzed for identification using Easy nLC1200/Q Exactive Plus. Separation was performed on a Waters ACQUITY UPLC Peptide BEH C18 column (2.1 × 100 mm, 1.8 μm). The mobile phase was made up of 0.1% formic acid (A) and acetonitrile (B). The elution gradient was set as follows: 0–3 min with 2–8% B; 3–42 min with 8–20% B; 42–48 min with 20–35% B; 48–49 min with 35–100% B; 49–60 min at 100 B%. The mass spectrometry conditions comprised an ESI+ detection mode with data-dependent scanning and full-scan acquisition (m/z 200–1600) in an orbital trap with a resolution of 70,000 (AGC 3e6). The parent ions of the first 20 isolated peptide signals (charge states ≥ +1) were fragmented via high-energy collision (HCD) with a normalized collision energy (NCE) of 28.0. The capillary’s temperature was 275 °C and the spray’s voltage was 1800 V. The maximum filling times were set to 50 ms and 45 ms for the full and MS-MS scans, respectively, and the dynamic exclusion time was set to 30 s.

Glutenin analysis was performed by crushing one wheat seed, and a glutenin extraction method was performed with slight modifications [51 ]. The extracted glutenin was analyzed using an UPLC system (Alliance e2695, Waters Corp., MA, USA) with an ACQUITY UPLC Peptide BEH C18 column (300A, 1.7 μm, 2.1 mm × 50 mm) and a photodiode array detector. The mobile phases were H₂O containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B). The injection volume of the dissolved samples was 3 μL, and the flow rate was 0.55 μL/min. The solvent gradient was changed from 21% (B) to 47% (B) from 0 to 30 min, and the column and sample temperatures were set to 55 °C and 10 °C, respectively.

Kinetic parameters for WT and variant SrtA were determined with the previously reported methods35 (link). SrtA catalyzed reaction between Abz-LPETGK(Dnp)-CONH₂ and GGG-COOH was measured at 37 °C with reaction time varied from 90 seconds to 15 minutes in buffer 30 mM Tris-HCl, 15 mM NaCl, 5 mM CaCl₂, pH 7.4. The SrtA concentration varied from 25 nM to 1000 nM. To determine K_cat and K_mLPETG, the concentration of GGG-COOH peptide was settled at 10 mM with Abz-LPETGK(Dnp)-CONH₂ concentration range from 100 μM to 8 mM. To determine K_mGGG, the concentration of Abz-LPETGK(Dnp)-CONH₂ was settled at 1 mM with GGG-COOH concentration range from 100 μM to 20 mM. The reaction was terminated by addition of 1/2 volume of 1 M HCl into the reaction mixture. The mixture was then subjected to UPLC system (ACQUITY UPLC Peptide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm ∗ 150 mm, Waters). The extent of the reaction was determined by measuring the absorbance of Dnp moiety at 355 nm. Reaction velocity was represented by enzyme turnover per second and fitted to Michaelis-Menten equation with OriginPro 8.5 software to determine parameters. Error bars were determined from at least three individual experiments.

Glutenin analysis was performed by crushing single grains of wheat, and the glutenin extraction method was performed as described previously [42 (link), 43 (link)]. The extracted glutenin was analyzed using a UPLC system (Alliance e2695, Waters Corp., MA, USA) with an ACQUITY UPLC Peptide BEH C18 column (300A, 1.7 μm, 2.1 mm × 50 mm) and a photodiode array detector. The mobile phases were H₂O containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B). The injection volume of the dissolved samples was 3 μL and the flow rate was 0.55 μL/min. The solvent gradient was changed from 21 to 47% (B) from 0 to 30 min, and the column and sample temperatures were set to 55 °C and 10 °C, respectively.

Lycopene was detected and measured using an Agilent LC system with UV/Vis diode array detector. Absorbance at 450 nm and 471 nm were monitored and the peak area corresponding to each component integrated to provide a measure of abundance. The LC column used was an Acquity UPLC Peptide BEH C18 column (2.1 × 100 mm, 1.7 μm, 300 Å, Waters). LC buffers were 50% (v/v) methanol in water (A) and 25% (v/v) ethyl acetate in acetonitrile (B). All solvents used were HPLC grade. The LC method was 6.5 min in total (0–1 min: 30% A, 70% B; 1–6 min: 0.1% A, 99.9% B; 6–6.5 min: 30% A, 70% B; at a flow rate of 0.3 ml/min) with 1.5 min of post-run time. The injection volume for the samples was 1.0 μl. Commercially available Lycopene (Sigma-Aldrich) was dissolved in acetone as a standard and a standard curve was generated.