Magnetic MXene with dual Ti-affinity sites for enhanced phosphopeptide enrichment in saliva

Saliva contains low-abundance phosphopeptides carrying important pathological information, but determining them with mass spectrometry is difficult due to matrix interference and low ionization efficiency. A well-designed nano-trapper with multiple affinity sites can help solve this problem. In this study, we developed a new dual Ti-affinity magnetic MXene (dTi-MMX). It combines the natural Ti-OH sites of MXene with additional Ti-O sites from titanium dioxide nanoparticles coated onto magnetic Ti₃C₂Tₓ MXene. This dual Ti-site approach significantly improves phosphopeptide capture, while the magnetic core allows for quick separation. Performance tests showed that dTi-MMX detects as low as 10 fmol‧µL^-1 of β-casein digests and selectively enriches phosphopeptides in mixtures of β-casein and BSA (at a 1:1000 mass ratio). When applied to human saliva, dTi-MMX successfully identified 110 unique phosphopeptides. Gene ontology analysis suggests that these peptides primarily participate in pathways such as salivary secretion and multicellular organismal processes, including several biomarkers associated with diseases, such as CST1 for cancer and LPO/CST3 for Alzheimer disease. This approach offers an effective tool for salivary phosphoproteomics research.