Synthesis of Polyethylenimine Functionalized Mesoporous Silica for In-Pipet-Tip Phosphopeptide Enrichment.

Synthesis of functionalized mesoporous silica material with large particle size remains a chanllenge. In this work, polyethylenimine (PEI) functionalized mesoporous silica (PFMS) with particle size as large as 100 μm was successfully synthesized by a facile method. In the synthesis process, PEI served as four roles simultaneously, including functionalized reagent, alkaline catalyst, template for particle formation, and pore-structure-directing agent. The surface areas of the products were higher than 260 m2/g. Benefiting from the large particle size and high surface area, PFMS was packed in a pipet tip to fabricate a convenient and miniaturized solid phase extraction apparatus for sample preparation. Additionally, based on the extremely abundant basic sites in the organic units of PFMS, the in-pipet-tip system was used as an anion-exchanger for phosphopeptide enrichment. The specificity of the developed method was investigated by capture of phosphopeptides from tryptic digests of standard protein mixtures, tryptic digests of nonfat milk, and human serum. Furthermore, the method was utilized to analyze phosphopeptides in tryptic digests of rat brain lysate, and 2251 unique phosphopeptides were successfully detected.