Waters e2695 system

LC–MS analysis of maker compounds in TH extract was conducted with a Waters e2695 system (ACQUITY QDa. Waters Corporation, Milford, MA) and Sunfire C18 (250 × 4.6 mm, 5 μm) column. Acetonitrile (A) and water with 0.1% formic acid (B) were used as mobile phases; the flow rate was 0.3 mL/min and the injection volume was 10 μL. Gradient program conditions were 0 min, 5% B; 0–30 min, 5–50% B; 30–60 min, 50–95% B. MS conditions were as follows: ESI mode, the capillary voltage was set to 0.8 kV, and the cone voltage was 15 V (+) or 30 V (−). MS data were acquired in the positive scan mode (mass range m/z 100–600 Da).

Theanine was extracted with distilled water as previously described, with some modifications (Tsushida and Takeo, 1984 (link)). About 100 mg of freeze-dried sample powder were dissolved in 3 ml distilled water and heated in a water bath at 100°C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatant was filtered with 0.22 μm filter for subsequent HPLC-based analysis of theanine content.
The detection conditions of HPLC analysis were as previously described (Dong et al., 2020 (link)). Theanine content was detected using the HPLC analysis (Waters e2695 system consisting a 2489 ultraviolet (UV)-visible detector, Waters Corporation, Milford, MA, United States), equipped with a C18 column (5 μm, 250 mm × 4.6 mm) at 28°C. The mobile phase comprised water (A), acetonitrile (B), and the gradient elution was performed as follows: B 0% (v/v) to 100% at 40 min, to 100% at 45 min and to 0% at 60 min. The amount of theanine was determined according to a calibration curve of theanine standard. L-Theanine standard and acetonitrile were purchased from Sigma-Aldrich (St Louis, MO, United States).

The customized anti-PD-L1 Adnectin ADX_5322_A02 was prepared by ChinaPeptides Co. Ltd (Shanghai, China). The sequencing report, protein expression, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis are shown in Additional file 1: Figs. S1, S2. Maleimide-NODA-GA was purchased from CheMatech (France), and high-purity hydrochloric acid from Merck (Darmstadt, Germany). Other chemicals were purchased from Sigma-Aldrich. The ⁶⁸Ga/⁶⁸Ge generator was purchased from ITG Isotope Technologies Garching Gmbh (Germany). PD-10 columns were purchased from GE Healthcare (Chicago, USA). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a Microflex LT/LRF system (Bruker Daltonics, Billerica, MA, USA). High-performance liquid chromatography (HPLC) /size exclusive chromatography (SEC) was performed on a Waters e2695 system equipped with a Superdex 200 Increase 10/300 GL column (GE Healthcare). Instant thin-layer chromatography (iTLC) was performed on Eckert & Ziegler Mini-Scan/FC (Hopkinton, MA, USA). The samples for binding affinity and biodistribution were measured with a gamma counter (Wizard 2480, Perkin Elmer Instruments Inc, Connection, USA).

Ten berries were selected from the three top, middle, and bottom bunches, and pooled. Berry pulps were ground under liquid nitrogen prior to further use. Each treatment was replicated three times. The extraction of soluble sugars was performed and determined via high-performance liquid chromatography (HPLC) as described by Zha et al. [14 (link)]. Soluble sugars were extracted from 3 g of frozen berry powder homogenized in 6 mL ethanol/water (4:1 v/v) at 35 °C for 20 min. The homogenate was centrifuged at 6500× g for 15 min, and the residues were re-extracted using the same procedure. The supernatants from the two extractions were mixed and made up to 15 mL with distilled water. Thereafter, 1 mL of the extract was evaporated under vacuum at 35 °C, redissolved in 1 mL MilliQ water (MilliporeSigma, Burlington, MA, USA), and passed through a 0.45 μm Millipore filter. Soluble sugar contents were determined using the Waters E2695 system (Waters, Milford, MA, USA).

The concentrations of vanillin and vanillyl alcohol were determined by a HPLC Waters system e2695 (Waters, USA) equipped with an Xbridge^TM-C18 column (Waters, USA). The peaks were detected at room temperature using ultraviolet detector (PDA-2998) at 210 nm with a mobile phase containing 40% absolute methanol (Chromatographic grade, Fisher Chemical, USA) supplied at a flow rate of 0.6 mL min⁻¹ [12 (link)].

The concentrations of extracellular vanillin and vanillyl alcohol were measured by an HPLC Waters system e2695 (Waters, USA) prepared with an Xbridge™-C18 column (Waters, USA). The peaks were detected at room temperature by an ultraviolet detector (PDA-2998) at 210 nm with a mobile phase of 40% absolute methanol (Chromatographic grade, Fisher Chemical, USA) supplied at a flow rate of 0.6 ml min^–1.