Synthesis and evaluation of superparamagnetic silica particles for extraction of glycopeptides in the microtiter plate format

Solid-phase extraction of glycopeptides (SPEG) coupled with quantitative proteomic analysis using mass spectrometry has shown great potential for investigating glycoproteins in an effort to discover new diagnostic biomarkers or therapeutic targets. As a solid-phase approach, SPEG can be performed with a microtiter plate to provide a high-throughput platform for large-scale screening of clinical samples. Here we describe the synthesis of superparamagnetic silica particles with hydrazide groups on the surface and further evaluate their use as the solid support for SPEG. We produced nonspherical silica particles containing superparamagnetic iron oxide cores using a modified Stober method and then derivatized their surface with hydrazide-terminated silane. Such composite particles displayed a strong response to the external magnetic field, and this feature enabled us to capture and release the particles easily for automated, high-throughput sample preparation of glycopeptides. When measured with standard glycoproteins, the adsorption capacity of these particles was >36 mg of glycoproteins per g of nanoparticles. The nanoparticles were used in a microtiter plate format for glycopeptide capture using a liquid handler. The captured glycopeptides were then analyzed by LC-MS and LC-MS/MS to determine the specificity and reproducibility of glycopeptide isolation. Our results demonstrate the potential of these superparamagnetic colloidal particles for high-throughput analysis of glycoproteins.