Sep pak c18 spe cartridge

Each isolated retina was prepared first by incubating in 200 µL 8 M aqueous urea solution for 30 min followed by centrifugation for 5 min at 1400× g [20 (link),23 (link)]. The supernatant was collected, and protein content was measured using a microplate reader (BioTek Synergy H1 with Take3 plate; Agilent, Santa Clara, CA, USA). An aliquot containing 100 µg protein was taken and the volume was adjusted with 25 mM ammonium bicarbonate to 100 µL. The sample was then reduced with dithiothreitol and carbamidomethylated with iodoacetic amide [20 (link),23 (link)]. After 9-fold dilution of the sample with aqueous 25 mM ammonium bicarbonate, trypsin digestion was performed at 37 °C overnight, then the reaction was quenched by acidification with formic acid (1 µL). The solution was desalted by solid-phase extraction (SPE) using 1 mL Sep-Pak™ C18 SPE cartridges (Waters, Milford, MA, USA), and the extract was dried under vacuum (Vacufuge™, Eppendorf AG, Hamburg, Germany) into a 1.5 mL centrifuge tube.

Saikosaponin A (SSa) and saikosaponin D (SSd) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Saikosaponins C (SSc), I (SSi), H (SSh), G (SSg), B1 (SSb₁), and B2 (SSb₂) were purchased from Chengdu Desite Biological Technologies Co., Ltd. (Chengdu, China). Their structures are shown in Figure 1. 15 batches of XG were supplied by Guangzhou Baiyunshan Guanghua Pharmaceutical Co., Ltd. (Guangzhou, China). Sep-Pak C18 SPE cartridges were obtained from Waters (Milford, MA, USA). LC-MS grade acetonitrile and acetic acid were purchased from Fisher (Loughborough, UK). Other reagents were of analytical grade and purchased from Titan Scientific Co., Ltd. (Shanghai, China). Deionized water was prepared by Milli-Q system (Bedford, MA, USA).

In-gel tryptic digestion [58 (link)] was carried out by dividing each lane of the gel into 4–5 equal parts and dicing them, followed by reduction (10 mM dithiothreitol in 100 mM ammonium bicarbonate), alkylation (55 mM iodoacetamide in 100 mM ammonium bicarbonate) and digestion with 13 ng/µL trypsin (Promega, Mannheim, Germany) in 10 mM ammonium bicarbonate containing 10% (v/v) acetonitrile. Tryptic peptides were extracted with a 1:1 mixture of 5% formic acid and acetonitrile and were completely lyophilized. The peptides were resuspended in 40 µL 0.1% formic acid prior to LC-MS/MS analysis. For the time-course analysis of the fungal supernatants from SCB or MZ substrates at 7, 14, 21, and 28 days, proteomic samples were obtained by in-solution digestion. Approximately 20 µg of protein was mixed with 6 M urea in 100 mM NH₄HCO₃ followed by reduction (100 mM DTT) and alkylation (300 mM iodoacetamide) and digestion (0.25 μg/μL trypsin). Samples were cleaned using Sep-Pak C18 SPE cartridges (Waters, Milford, MA, USA). After lyophilization, the tryptic peptides were resuspended in 20 µL 0.1% formic acid prior to LC-MS/MS analysis.

Aqueous extracts from the roots were purified using Sep-Pak C18 SPE cartridges (360 mg sorbent per cartridge, 55–105 µm) from Waters (Milford, MA, USA). Prior to use, the cartridges were activated with 96% ethanol and rinsed with water. A diagram of fructan purification and fractionation is presented in Figure 1.
For purification, 5 mL aliquots were slowly loaded into a cartridge and then it was washed with 2 mL distilled water to elute any non-retained material. Afterwards, the cartridge was washed with 5 mL of 20% ethanol. Water and 20%-ethanol fractions were analyzed for fructan content.
For fructan fractionation, the water-eluted fraction was freeze-dried and redissolved in 1 mL water in a screwcap tube. 4 mL 96% ethanol were added and the samples were left overnight at 4 °C. The ethanol-insoluble material was recovered by centrifugation, and the ethanol-soluble fraction was concentrated, lyophilized and assayed for fructan content (Fruct1). The fructan content of the ethanol-insoluble fraction (Fruct2) was calculated by subtracting the fructans present in Fruct1 from the total quantified in the water-eluted fraction. The fructans present in the 20% ethanol-eluted fraction were called Fruct3, as illustrated in Figure 1.

According to the traditional concentration and enrichment methods for MCs, typical MCLR-DBPs were purified [25 (link),26 (link)]. Disinfection samples (50 mL) were applied to the pre-rinsed SepPakC₁₈ SPE cartridges (1000 mg, Waters) with 10 mL methanol and 10 mL high purity water. Impurities and typical MCLR-DBPs were eluted with 5 mL 10% methanol and 5 mL 80% methanol, respectively. The crude extracts of typical MCLR-DBPs were evaporated to dryness in N₂ flow and resuspended in 200 µL 20% acetonitrile. Then the crude extracts were separated using a Dionex Ultimate-3000 HPLC system equipped with a C₁₈ reversed-phase preparative column (25.4 mm × 450 mm, 5 µm, 120 Å) [21 (link)]. Water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) were used as mobile phase A and mobile phase B, respectively. The elution conditions were: 20% mobile phase B for 3 min; 20%→80% mobile phase B over 25 min; 80%→20% mobile phase B within 0.1 min; 20% mobile phase B for 3 min. The column temperature and the flow rate were set at 35 °C and 5 mL/min, respectively.

Samples were re-suspended in 350 μL 8 M urea/ 100 mM Tris HCl pH 8.5 after TCA/acetone precipitation and phenol extraction. Briefly, samples were reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) for 30 min at room temperature and alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark. Then, proteins were firstly digested for 5 h at 37°C with 250 ng rLys-C Mass Spec Grade (Promega, Madison, USA). Samples were then diluted 4-fold with 100 mM Tris HCl pH 8.5 to reach a concentration of 2M urea and then re-incubated overnight at 37°C with 1 μg Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA). A second incubation with the same amount of trypsin (5 h at 37°C) was performed to ensure a complete digestion. Digestion was stopped by adding formic acid to 5% final concentration and peptides were desalted and concentrated on Sep-Pak C₁₈ SPE cartridge (Waters, Milford, MA, USA) according to manufacturer’s instructions.