Coupling of Phosphate-Imprinted Mesoporous Silica Nanoparticles-Based Selective Enrichment with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry for Highly Efficient Analysis of Protein Phosphorylation.

Protein phosphorylation is a major post-translational modification and represents a ubiquitous mechanism for the cellular signaling of many different biological processes. Selective enrichment of phosphopeptides from the complex biological samples is a key step for the mass spectrometric (MS) analysis of protein phosphorylation. Herein, we present phosphate-imprinted mesoporous silica nanoparticles (MSNs) as an ideal sorbent for selective enrichment of phosphopeptides and an off-line combination with matrix-asisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for highly efficient analysis of protein phosphorylation. The phosphate-imprinted MSNs were prepared according to a newly reported strategy called dual-template docking oriented molecular imprinting (DTD-OMI). The prepared molecularly imprinted mesoporous material exhibited several significant merits, such as excellent selectivity toward phosphopeptides, tolerance to interference, fast binding equilibrium, and large binding capacity, which made the molecularly imprinted mesoporous material an ideal sorbent for selective enrichment of phosphopeptides. Using β-casein as a representative phosphoprotein, highly efficient phosphorylation analysis by the off-line platform was verified. Phosphorylation analysis of a nonfat milk sample was also well demonstrated. Because of their highly desirable properties, the phosphate-imprinted MSNs could find more applications in the analysis of protein phosphorylation.