Experimental Technology and Management

[Objective] Efficient enrichment of phosphopeptides is crucial for phosphoproteomics research, enabling the identification of disease biomarkers and advancing early clinical diagnostics. Two-dimensional transition metal carbides(MXenes), particularly Ti_3C_2T_x, have attracted considerable attention for protein separation due to their large specific surface area and abundant surface functional groups. However, conventional methods for preparing TiO₂/Ti_3C_2T_x composites suffer from weak interfacial bonding and insufficient control over the TiO₂ crystal phase, both of which critically influence phosphopeptide enrichment performance. To address these limitations, this study developed a green and controllable solvent-engineering strategy for the in situ preparation of TiO₂/Ti_3C_2T_x heterostructures with tailored crystal phases to achieve efficient phosphopeptide enrichment. [Methods] Few-layer Ti_3C_2T_x MXene was first synthesized from Ti₃ AlC₂ using the minimally intensive layer delamination method with LiF/HCl etching. Thereafter, TiO₂/Ti_3C_2T_x heterostructures with controlled crystal phases were prepared via solvothermal treatment of the Ti_3C_2T_x precursor in different solvents(ethylene glycol, ethanol, and deionized water), denoted as TiO₂/Ti_3C_2T_x-G, Ti O₂/Ti_3C_2T_x-E, and TiO₂/Ti_3C_2T_x-H, respectively. The resulting composites were systematically characterized using SEM, TEM, XRD, and XPS, and their phosphopeptide enrichment performance was evaluated using model proteins analyzed by MALDI–TOF mass spectrometry. [Results] SEM, TEM, and XRD characterizations revealed that TiO₂/Ti_3C_2T_x-G maintained the MXene sheet structure with sparse, small anatase TiO₂ nanoparticles; TiO₂/Ti_3C_2T_x-E exhibited uniformly distributed anatase TiO₂ nanoparticles with diameters of 10–30 nm; and TiO₂/Ti_3C_2T_x-H possessed a unique bi-modal size distribution of TiO₂ nanoparticles and polyhedra with a mixed phase of anatase and rutile. XPS analysis further demonstrated a progressive increase in Ti(Ⅳ) content from the ethylene glycol to the water system. These results confirmed the successful formation of TiO₂/Ti_3C_2T_x heterostructures with solvent-dependent morphologies and crystal phases. The solvent-dependent properties of TiO₂/Ti_3C_2T_x heterostructures were attributed to the different oxidation kinetics dictated by solvent oxidizability. In the enrichment experiments, direct analysis of the protein digest identified only a few phosphopeptides. Treatment with all Ti O₂/Ti_3C_2T_x heterostructures significantly improved phosphopeptide enrichment and showed distinct performance. In particular, TiO₂/Ti_3C_2T_x-G, Ti O₂/Ti_3C_2T_x-E, and TiO₂/Ti_3C_2T_x-H captured 16, 31, and 35 distinct phosphopeptides, respectively. The mixed-phase TiO₂/Ti_3C_2T_x-H composite exhibited the highest enrichment efficiency. This superior performance was attributed to the synergistic effect of the high concentration of Ti(Ⅳ) sites in well-crystallized TiO₂, which enabled specific Ti—O—P coordination, and the residual MXene substrate, whose surface functional groups facilitated interactions with phosphopeptides via hydrogen bonding. [Conclusions] This study successfully developed a green and efficient solvent-engineering strategy for the controllable preparation of TiO₂/Ti_3C_2T_x heterostructures with tailored crystal phases and morphologies without requiring external titanium sources. The results demonstrated that the crystal phase and surface chemistry of these heterostructures could be precisely modulated by the solvothermal solvent, determining their phosphopeptide enrichment performance. The mixed-phase anatase/rutile TiO₂ grown in water exhibited the highest efficiency, capturing the most phosphopeptides. This work provides a novel and sustainable pathway for fabricating MXene-based functional heterostructures and establishes their considerable potential as high-performance platforms for phosphoproteomics analysis. The insights into the structure–property relationship offer a valuable design principle for developing advanced separation materials, with potential applications in energy storage and catalysis.