Tsq altis triple quadrupole mass spectrometer

A Dionex Ultimate 3000 RS UHPLC + focused system coupled with a TSQ Altis triple quadrupole mass spectrometer (MS/MS, Thermo Fisher Scientific, Austin, TX, USA) was used to detect residues of the seven neonicotinoids. Trace Finder software (version 4.1) was used for analysis, data acquisition, and reporting. An Accucore RP-MS C18 column (150 × 2.1 mm, 2.6 μm film thickness, Thermo Fisher Scientific) was used for separation at 40 °C. Mobile phase A was water, and mobile phase B was methanol; the flow rate was 0.30 mL/min. The gradient program of the mobile phase was 0–2 min 10% B, 2–6 min from 10% B to 90% B, 6–8 min 90% B, 8–8.1 min from 90% B to 10% B, and 8.1–16 min 10% B. The injection volume was 2 μL. To optimize the MS/MS parameters of the tested analytes (Table S1), a Harvard infusion pump (Harvard Apparatus, South Natick, MA, USA) was used. The precursor ions [M + H]⁺ were identified in the multiple reaction monitoring (MRM) mode using an electrospray ionization interface in the positive ion mode (H-ESI+). The MS conditions were as follows: the ion source temperature was set at 325 °C, the ion spray voltage was set at 3800 V, and the sheath and auxiliary gasses were 40 and 10 arb, respectively. Trace Finder (version 4.2) software was used for data acquisition.

Vanquish Flex Ultra-performance Liquid Chromatography (UPLC) and TSQ Altis Triple Quadrupole Mass Spectrometer (MS) (Thermo Fisher Scientific, United States), nitrogen blowing concentrator (Beijing Politech Instrument Co., Ltd., China), High-speed Refrigerated Centrifuge (Yancheng Kait Experimental Equipment Co., Ltd. China), Vortex Meter (Wiggens, Germany), Milli-Q Purification System (Millipore, United States), KQ-500E Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd. China), and Electronic analytical balance (Mettler Toledo, United States).

The transition list of differentially expressed proteins was prepared using Skyline^®. Transitions of specific proteins were optimized along with the LC parameters. All runs were performed using nano-LC mode. The assay was performed in Triple Quadrupole Instrument (TSQ, ThermoFisher Scientific, United States) Peptides (1 μg) was reconstituted in 10 μl 0.1% FA. The peptides were then run in a gradient comprising of solvent A (0.1% FA) Solvent B (80% ACN with 0.1% FA) for 45 min per run. The flow rate was maintained at 300 nl/min and the column used was ES 803 Easy spray pepmap C18 column. The acquisition was done using TSQ Altis™ Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, United States) using a method duration of 45 min.

The TLS assay was previous described [12 (link)]. Briefly, 200 ng of competitor gapped plasmid and the 50 ng of lesion-containing plasmid were transfected into HT29 and SW480 cells using Lipofectamine2000 (ThermoFisher). Cells were incubated at 37 °C for 4 h and harvested. Subsequently, DNA was extracted using DNA isolation kit (QIAGEN) and transformed into the recA- E. coli to propagate closed plasmids for 16 h. Plasmid DNA was extracted from E. coli culture and lesion region was amplified by PCR (forward primer: 5′ TTGTACTGAGAGTGCACCATGCCCGT-3′, reverse primer: 5′-GAGTCAGTGAGCGAGGAAGCGTGCTG-3′). Restriction enzymes XhoI and SphI (NEB) were used to digest the PCR products, which were next subjected to TSQ Altis™ Triple Quadrupole Mass Spectrometer (ThermoFisher) to determine the nucleotides originally repaired in CRC cells.