Integrating proteomic and metabolomic profiles to develop a panel of salivary biomarkers for colorectal cancer detection

This study integrated the diagnostic potential of saliva with multi-omics approaches for CRC research. We employed Olink PEA and LC-MS/MS to analyze protein and metabolite changes in the saliva of CRC patients. Using machine learning, we developed a protein-metabolite biomarker model with good diagnostic performance, offering novel insights for CRC screening and potential intervention strategies.Relative quantification of 92 proteins in saliva supernatants from CRC patients and HC participants was achieved with the Olink Target 96 immuno-oncology panel (Olink Proteomics AB, Sweden). This panel utilizes PEA technology, enabling concurrent detection of 96 analytes (92 protein markers and 4 internal controls) from just 1 μL of a sample. The PEA technology involves paired oligonucleotide-labeled antibody probes binding to target proteins, and, upon addition of DNA polymerase, proximity-dependent DNA polymerization generates a unique PCR target sequence. The microfluidic qPCR instrument (Signature Q100, Olink Proteomics AB, Sweden) was used to detect and quantify this DNA sequence.LC-MS/MS analysis was conducted using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) coupled with a UHPLC system (Dionex Ultimate 3000, Thermo Fisher Scientific, USA). Separation was achieved on a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters, USA) at 35°C, with an autosampler temperature of 4°C and an injection volume of 4 μL. In positive ion mode, mobile phase A was distilled water (Watsons, China) with 10 mM ammonium formate (LC-MS grade, Sigma-Aldrich, USA) and 0.1% formic acid (LC-MS grade, Aladdin, China), while mobile phase B was acetonitrile (LC-MS grade, Thermo Fisher Scientific, USA) containing 0.1% formic acid. For negative ion mode, mobile phase A was distilled water with 10 mM ammonium formate, and mobile phase B was acetonitrile. The flow rate was maintained at 0.3 mL/min with the following gradient: 0 min, 2% B; 0–1 min, 2% B; 8 min, 98% B; 8–10 min, 98% B; 10.5 min, 2% B; 10.5–12 min, 2% B. ESI source parameters were set with a sheath gas flow of 50 arb, auxiliary gas flow of 15 arb, and capillary temperature of 320°C. Data acquisition used data dependent acquisition (DDA) mode, covering a scan range of 80–900 m/z, with MS and MS/MS resolutions of 60,000 and 15,000, respectively, normalized collision energies (NCE) of 20/30/40, an 8 s dynamic exclusion, and spray voltages of 3.5 kV (positive) and -3.2 kV (negative). QC samples were analyzed under the same conditions as regular samples, with one QC sample injected after every six samples to ensure analytical stability.