In-Tip Nanoreactors for Simultaneous Proteolysis and Enrichment of Phosphorylated Peptides

Protein phosphorylation introduces negative charges on the hydroxyl groups of serine, threonine, and tyrosine residues, reducing the ionization efficiency of phosphorylated peptides. The low abundance of phosphorylated peptides often diminishes their detection using mass spectrometry. To enhance the identification of the low-abundance peptides, an enrichment step was often used, which complicated the high-throughput analysis of phosphorylated proteomes. In this study, we developed a titanium dioxide surface-modified macroporous silicon encapsulated micropipette tips, loaded with trypsin, to integrate rapid enzymatic protein hydrolysis with selective enrichment and extraction of phosphorylated peptides within a microfluidic enzyme reactor. This streamlined approach simplified the protein sample preparation process, combining enzymatic hydrolysis, selective enrichment and separation while maintaining high efficiency. The method enabled comprehensive analysis of complex cancer cell line samples in 1-2 h. Successful detection of phosphorylated peptides from protein mixtures was achieved using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry. This application may provide the potential for high-throughput phosphoproteomics and advance the study of protein modifications.