Packed in-tube solid-phase microextraction with mesoporous zirconium titanium oxides for highly efficient phosphopeptide enrichment and analysis

Phosphorylated peptides are crucial for understanding biological regulation processes. However, the detection of phosphorylated peptides remains challenging in mass spectrometry (MS) due to their high hydration, low abundance, and the significant signal suppression caused by non-phosphorylated peptides. It is essential to enhance the efficiency of phosphopeptide enrichment from complex biological samples to achieve comprehensive analysis and identification of phosphopeptides. Herein, a packed in-tube solid-phase microextraction (IT-SPME) method was developed based on mesoporous zirconium titanium oxides for the selective enrichment and analysis of phosphopeptides. The IT-SPME system achieved the efficient capture of phosphopeptides from complex biological samples followed by high-sensitivity detection (5 fmol). This exceptional performance can be attributed to the high surface area (289.77 m2/g) of mesoporous zirconium titanium oxides, which provided abundant active sites for a high loading capacity. The zirconium titanium oxides with a mesoporous structure (∼4.5 nm) was prepared through a sol–gel and solvothermal process. Importantly, this method proved effective in enriching phosphopeptides from practical samples such as non-fat milk and egg white. It is the first attempt to employ a mesoporous packed IT-SPME approach for phosphopeptide enrichment. These results demonstrate the significant application potential of mesoporous zirconium titanium oxides in phosphoproteomics.