Tripletof 6600 system

Metabolic profiling of samples was performed on an Agilent 1290 Infinity LC system (Agilent Technologies, Santa-Clara, California, USA) coupled with an AB SCIEX Triple TOF 6600 System (AB SCIEX, Framingham, MA, USA) in Shanghai Applied Protein Technology Co., Ltd.

Approximately 100 mg stool was extracted with 1 ml methanol, and 60 μL 2-Chloro-l-phenylalanine and Heptadecanoic acid were added as the internal standard. After vortex, ultrasonic treatment and centrifugation, the supernatant was transferred into a new tube and dried. Then 60 μL 15 mg/ml methoxyamine pyridine and N, O-Bis(trimethylsilyl)trifluoroacetamide reagent were sequentially added and incubated at 37°C. After centrifugation, the supernatant was analyzed using the ACQUITY UPLC system (Waters Corporation, Milford, United States) coupled with AB SCIEX Triple TOF 6600 System (AB SCIEX, Framingham, MA) in positive and negative ion modes according to the manufacturer’s manual (Yan et al., 2020 (link)).

Approximately 100 mg stool or colonic tissue was extracted with 1 mL methanol, and 60 µL 2‐Chloro‐L phenylalanine and Heptadecanoic acid was added as the internal standard. After vortexing, ultrasonic treatment and centrifugation, the supernatant was transferred into a new tube and dried. Then 60 µL 15 mg mL⁻¹ methoxyamine pyridine and N,O‐Bis(trimethylsilyl)trifluoroacetamide reagent was sequentially added and incubated at 37 °C. After centrifugation, the supernatant was analyzed using the ACQUITY UPLC system (Waters Corporation, Milford, USA) coupled with AB SCIEX Triple TOF 6600 System (AB SCIEX, Framingham, MA) in positive and negative ion modes according to the manufacturer's manual. Metabolomic data were analyzed with MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml).

Fasting serum samples of each patient with UC at Phase 2 were collected and preprocessed. Ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry was performed on an Agilent 1290 Infinity LC system (Agilent Technologies, Santa-Clara, California, USA) coupled with an AB SCIEX Triple TOF 6600 System (AB SCIEX, Framingham, MA, USA) in Shanghai Applied Protein Technology Co., Ltd. (China). Both positive and negative ion modes were considered.

The chromatographic system, which was set up in accordance with the method described previously [10 ] with modifications, which consisted of a Shimadzu LC-30A UHPLC system equipped with an ultraviolet detector. Chromatographic separation was achieved using a Waters ACQUITY UPLC™ BEH C18 (2.1 × 100 mm, 1.7 μm) at a flow rate of 0.3 ml/min. The column oven temperature was set at 40 °C. The mobile phase was a binary solvent system consisting of solvent A (formic acid 0.1% [v/v] in water) and solvent B (acetonitrile). The gradient conditions were as follows: 5–15% B from 0 to 25 min, maintained at 15% B for 20 min; 15–18% B from 45 to 60 min; 18–95% B from 60 to 70 min, maintained at 95% B for 2 min; and 5% B from 72 to 75 min for equilibration of the column for the next run. The sample injection volume was 3 μL. Chromatograms were recorded at 520 nm for anthocyanins. The analytes were identified using an AB SCIEX TripleTOF® 6600 system equipped with a Turbo V™ ion source. Product ion spectra were acquired using time-of-flight mass spectrometry and information-dependent acquisition compound scan modes. The data acquisition used Analyst® TF 1.7.1 software, and PeakView® 2.1 and MasterView™ software were used for data analysis.

The full-length sequence of circAnks1a was constructed into a linear DNA template containing the T7 promoter. A biotinylated RNA probe was transcribed in vitro using T7 RNA polymerase (Thermo Scientific) and bound to streptavidin C1 magnetic beads (Invitrogen). Spinal dorsal horn tissues were ground in liquid nitrogen and incubated in lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl₂, 1% NP40, SUPERase-In (Ambion), and protease inhibitors (Roche)] on ice for 30 min. The lysates were then incubated with the RNA probe for 2 h at 4 °C. The beads were briefly washed five times with wash buffer and boiled in sodium dodecyl sulphate (SDS) buffer. The retrieved proteins were separated via SDS-PAGE. The gel bands were manually excised and digested with mass spectrometry-grade trypsin (Promega). The digested peptides were analyzed on an AB Sciex TripleTOF® 6600 System (AB Sciex). The mass spectrometry data were analyzed and identified using Mascot (Matrix Science) in the NCBI Rattus database and the UniProt Rattus database.