Fabrication of functionalized SiO2/TiO2 nanocomposites via amidation for the fast and selective enrichment of phosphopeptides

Abstract

Singly-functionalized nanomaterials cannot fully meet the requirements for biological applications, although most of them have still been used in the enrichment of phosphopeptides. Multifunctional nanocomposites with more capacity for the specific enrichment of phosphopeptides are becoming a new research focus. Here, a simplified and effective system was developed for the fast and highly selective enrichment of phosphopeptides using functionalized SiO₂/TiO₂ nanocomposites as sorbents. The functionalized SiO₂/TiO₂ nanocomposites were prepared by an amidation reaction between the amino groups of mesoporous SiO₂ nanoparticles and the carboxyl groups of TiO₂ nanocrystals. The nanocomposites' surfaces possess high absorption capacity for phosphopeptides, taking advantage of both the strong anion exchange ability of the amine-functionalized SiO₂ nanospheres and the high metal oxide affinity of the TiO₂ nanocrystals. A simplified approach using a pipette tip filled with prepared functionalized SiO₂/TiO₂ nanocomposites was applied to enrich phosphopeptides in digests of phosphoprotein (β-casein), mixtures of β-casein and bovine serum albumin (BSA), and non-fat milk. Using this approach, enriched phosphopeptides were obtained from the digested peptides in less than one minute. The phosphopeptides were analyzed by MALDI-TOF-MS, and the limit of detection for phosphopeptides was approximately 10 femtomoles. With highly selective enrichment efficiency, the functionalized SiO₂/TiO₂ nanocomposites could have great promise for applications in MS-based phosphoproteomics.