Distinctive Lipid Characteristics of Colorectal Cancer Revealed through Non-Targeted Lipidomics Analysis of Tongue Coating

Tongue coating was used as a sample in this study.Samples were obtained in the morning on an empty stomach. Prior to tongue coating collection, participants rinsed their mouths with sterile water, then used a sterile cotton swab to collect tongue coating. The cotton swab head was placed into a 1ml cryotube containing sterile water for rinsing, then discarded after. The cryotube was swiftly quenched in liquid nitrogen and then stored at -80℃.Lipidomic analyses utilized an Ultimate 3000 UHPLC system coupled with Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific,USA). A reversed-phase CSH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters) was maintained at 45℃. Samples, re-suspended in 2-isopropanol: acetonitrile: water (30:65:5, v/v/v), were injected (2 μL). In positive ion mode, mobile phase A was acetonitrile: water (60:40, v/v) with 10 mM ammonium formate and 0.1% formic acid, and B was 2-propanol: acetonitrile (90:10, v/v) with 10 mM ammonium formate and 0.1% formic acid. In negative ion mode, A was acetonitrile: water (60:40, v/v) with 10 mM ammonium formate, and B was 2-propanol: acetonitrile (90:10, v/v) with 10 mM ammonium formate. The flow rate was set to 0.4 mL/min with the following gradient: 0 min, 15% B; 2 min, 30% B; 3 min, 48% B; 14.5 min, 82% B; and 15 min, 99% B. The sample temperature was maintained at 10 ℃. Operational parameters: sheath gas pressure 40, auxiliary gas flow 15, sweep gas flow 0, spray voltage 3.2 kV (positive) and -3.5 kV (negative), capillary temperature 320 ℃, Aux gas heater temperature 350 ℃.  Data were acquired in the data-dependent acquisition mode, with each cycle comprising an MS1 at 70,000 resolution and an MS/MS scan at 17,500 resolution (120-1200 m/z range). The normalized collision energy was 20/30/40, and dynamic exclusion was 8 s. Acquisition was conducted in positive and negative ion modes separately. Automatic gain control targets were 1e6 for MS1 and 1e5 for MS/MS. Samples were randomly analyzed, with interspersed quality control (QC) samples analyzed after every 8 samples during lipidomic analysis. Raw data were collected using Xcalibur (V4.1,Thermo Fisher Scientific,USA).