Xb c18

Hungarian red sweet pepper powder (in 1–5 g) was applied in triplicates to extract carotenoids and extraction was carried out with 50 mL dichloroethane:acetone:methanol as solvent mixture in 2:2:1 ratio. Next, the mixture was mixed in an ultrasonic water bath for 30 min, then filtered through a filter paper (Munktell-292). Afterwards, a 0.22 µm PTFE syringe filter (TPP Techno Plastic Products AG, Trasadingen, Switzerland) was used for further purification. Filtered residue was vaporized at 40 °C at 0.2 bar and then filtrate was solved in an high-performance liquid chromatographic (HPLC) pigment reagent (isopropanol:ACN:methanol in 55:35:10 proportion) (Merck, Darmstadt, Germany) [50 (link)]. A HPLC separation was performed on Phenomenex Kinetex^® column (2.6 µm, XB-C18, 100 Å, 100 × 4.6 mm) (Phenomenex, Torrance, CA, USA) with 2 gradient elutions: A: 11% methanol, B: isopropanol:ACN:methanol (55:35:10 V/V/V%) mixture. Gradient elution steps were the following: 0–3 min solvent A 100%; 15–20 min solvent A 20%; 25–45 min solvent B 100%; 48–50 min solvent A 100%. Flow rate was 0.6 mL/min and Diode Array Detector (DAD) detection was carried out on 460 nm and 350 nm. Samples were injected in 10 µL and after DAD detection was applied at 460 nm and 350 nm. HPLC profile is shown in Figure 1. Carotenoid compounds with the greatest areas were identified and involved in Table 1.

Ubiquitin pepsine hydrolysate (1 mg, 5.78 × 10⁻⁷ mol) was dissolved in 1 mL of triethylammonium bicarbonate (TEAB) buffer (pH 8.4–8.6). The solution was divided into two portions: 0.5 mL was retained as a control sample and the second 0.5 mL was tagged with 1.13 mg (57 eq.) of 5a dissolved in 113 μL of TEAB and incubated for 1 h. Water (0.7 mL) was added to hydrolyze the unreacted active ester. Both samples of protein hydrolysates, derivatized and non-derivatized, were evaporated on a speedvac. Subsequently, they were diluted 50 times with water and analyzed by LC-MS. The LC-MS analysis was performed on micrOTOF-Q coupled with the analytic HPLC Agilent 1200, equipped with the UV detector—210 and 280 nm, RP column: Aeris Peptide, Phenomenex XB-C18 (50 × 2.1 mm, 100 Å, 3.6 μm), flow—0.2 mL/min, injection volume—5 μL, eluents: A = H₂O + 0.1% HCOOH, B = 80% MeCN in H₂O + 0.1% HCOOH with the gradient: 0–5% B in 3 min, 5–60% B in 55 min., 60–100% B in 40 min. The separation was performed at room temperature (22 °C).

The extraction and detection of erythrydiol (2) follow the procedure described for β-amyrin. For the rest of the triterpenoids, 200 µl of culture was collected in a microfuge tube before it was directly extracted with 800 µl methanol with bead-beating (3,800 rpm, 1 min × 2). The mixture was centrifuged at 12,000g for 1 min to separate the pellet. Two hundred microlitres of the supernatant was transferred into an Eppendorf tube, which was then evaporated in a vacuum concentrator at room temperature and the remainders were resuspended in 200 µl methanol. Finally, samples were filtered with Amicon Ultra 0.5-ml 3-kDa filter tubes or centrifuged at 15,000g for 5 min. Products were analysed using LC–MS (1260 Infinity II LC-MSD iQ, Agilent) equipped with a reverse phase C18 column (Kinetex 2.6 µm, 250 × 4.6 mm, XB-C18, Phenomenex). A 50-min isocratic method was performed with 10:90 of water (solvent A) and acetonitrile (solvent B) using a flow rate of 0.3 ml min⁻¹. Full mass spectra were generated for metabolite identification by scanning within the m/z range of 300–600 in negative-ion mode. Data acquisition and analysis were performed using OpenLab CDS version 2.4 (Agilent).