Symmetry c18 precolumn

To separate the digested samples, a Waters Acquity liquid chromatography M-Class system (Waters Corp., Milford, MA, USA) equipped with a Peptide BEH C18 analytical column (150 mm × 75 µm; 1.7 µm, Waters Corp.) and a Symmetry C18 precolumn (180 µm × 20 mm; 1.7 µm, Waters Corp.) was performed. Each sample was injected into the pre-column and washed with 99% solvent A (0.1% formic acid in water). Then, peptides were transferred to an analytical column. A mass spectrometry (MS) using Synapt G2-Si (Waters Corp.) with a nano-electrospray ionization (nESI) source operating under a positive ion mode was performed. Raw chromatography files were analyzed with Byonic software (Protein Metrics, Cupertino, CA, USA). The detected peptides were compared to the SWISSPROT dog proteome (CANLF) downloaded in February 2022. The total intensity was a sum of all the peak intensities over all MS/MS spectra. The protein p-value is the likelihood of the peptide-spectrum matches (PSMs) to this protein (or protein group) arising by random chance, according to a simple probabilistic model. A log p-value of −3.0 corresponds to a protein p-value of 0.001, or one chance in a thousand. A detailed method was described in our previous study [31 (link)].

Aliquots of tomato powders (0.25 g dw) were analyzed monthly up to 180 d, except for samples incubated at aw 0.56, which were analyzed up to 106 d. Extraction was performed with 10 mL of methanol. The mixture was vortexed for 1 min, mixed continuously for 30 min with a magnetic stirrer, and then centrifuged at 12.000× g and 5 °C for 10 min. Extractions were carried out in triplicate on initial samples and in duplicate for samples stored at different a_w levels. The phenolic contents of methanolic extracts were analyzed by HPLC as described previously [13 (link)]. A 250 × 4.6 mm i.d., 5 μm particle size, Symmetry reverse phase C-18 column (Waters) equipped with a Symmetry C-18 precolumn was used. Formic acid (5%) was added to both methanol and water before the following mobile phases were prepared: (A) water/methanol (95:5, v/v); (B) water/methanol (88:12, v/v); (C) water/methanol (20:80, v/v); (D) and methanol. The following gradient elution was used: 0−5 min, 100% A; 5−10 min linear gradient to reach 100% B; 10−13 min, 100% B; 13−35 min linear gradient to reach 75% B and 25% C; 35−50 min linear gradient to reach 50% B and 50% C; 50−52 min linear gradient to reach 100% C; 52−57 min, 100% C; 57−60 min, 100% D. The flow rate was 1 mL/min. Chlorogenic acid was quantified at 330 from calibration curves using pure standard and expressed as milligrams per kilogram of dry product.

LC-MS/MS analysis was performed on a nanoAcquity chromatography system (Waters Corporation, Milford, MA) coupled to a LTQ-Orbitrap Velos (Thermo Fisher Scientific, Waltham, MA). CSF digests were trapped onto a Symmetry C18 precolumn (180 μm id, 20 mm long, 5 μm particles) (Waters Corporation, Milford, MA) over 3 min, at a flow rate of 25 μL/min in 2% (v/v) ACN /0.1% (v/v) formic acid. Bound peptides were resolved on a nanoAcquity UPLC C18 column (75 μm id, 150 mm long, 3 μm particles) at 300 nL/min over a 120 min linear gradient from 3 to 85% (v/v) ACN in 0.1% v/v formic acid. The instrument was operated in data-dependent acquisition mode. A fixed volume (0.5 μL) of digestion was analysed and the 20 most intense multiply charged ions were sequentially fragmented at 35% of normalized collision energy. Precursors selected were dynamically excluded for 20 s.

The analysis was performed on a nanoACQUITY Ultra-Performance-LC (UPLC, Waters Corporation, Milford, USA). The samples were trapped on a 20 × 0.18 mm, 5 µm Symmetry C18 precolumn (Waters Corporation, Milford, USA), and the peptides were separated on a ACQUITY UPLC^® BEH130 C18 column (Waters Corporation, Milford, USA), 75 µm × 250 mm, 1.7 µm particle size. The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Trapping was performed during 3 min at 5 µL/min with 97% of solvent A and 3% of solvent B. Elution was performed at a flow rate of 300 nL/min, using 3–40% gradient (solvent B) over 79 min followed by 80% (solvent B) over 10 min at 60 °C.

Sample preparation: an aliquot of the culture liquid (0.25 mL) was diluted 10 times with a mixture of acetonitrile:isopropanol (50:45, v/v), mixed and centrifuged within 8 min at 12,100 × g. The supernatant was used for analysis. HPLC analyses were performed on an Agilent Infinity 1200 system (Agilent Technologies, Germany) with a Symmetry C18 column (4.6 × 250 mm, 5 μm) with a Symmetry C18 precolumn (3.9 × 20 mm, 5 μm,) (Waters, Milford, MA, USA) at 50 °C and a flow rate of 1 mL/min. Steroid assay was performed using two mobile phases: I—acetonitrile:2-propanol:water (50:45:5 (v/v/v)) and II—acetonitrile:water:TFA (60:40:0.02 (v/v/v)) with gradient elution as described [43 ], UV-detection at 200 (I) and 240 nm (II). Calibrations were performed by the external standard method based on peak areas. The results were processed using the ChemStation Rev. software. B.04.03 (Agilent Technologies, USA). Retention times (Rt) for mobile phase I: cholesterol, Rt 10.3; phytosterols, Rt 9.5–10; pregnenolone, Rt 3.16; for mobile phase II: progesterone, Rt 10.56. Rt of 3-methoxymethyl derivatives are given in Table 1.