Pipette-tip selective extraction of glycoproteins with lectin modified gold nano-particles on a polymer monolithic phase

摘要

The selective extraction of specific proteins (non glycosylated, glycosylated or different glycoforms) from complex sample matrices utilising selective solid phase extration (SPE) is of significant interest within the fields of proteomics and glycoproteomics. Polymer monoliths have proven to be an excellent solid support for SPE applications in bio-analysis due to their excellent mass transfer characteristics for large biomolecules. Although biorecognition molecules such as lectins can be covalently immobilised directly onto the 10 monolith surface using a variety of chemistries, the relatively low surface area of these monoliths results in a correspondingly low sample capacity. Recently, we have described the covalent attachment of 20 nm gold nanoparticles (AuNPs) upon a polymer monolith with excellent surface coverage leading to a significant increase in surface area. In this work therefore wedescribe the in-situ preparation of an ethylene dimethacrylate porous polymer monolith within the confines of 20 μL polypropylene pipette tips, onto which methacrylate anchor sites were previously grafted to ensure intimate monolith/wall contact. Then AuNPs were immobilised onto the monolith pore 15 surface utilising azlactone cemistry. Field emission scanning electron microscopy was used to verify the high surface coverage of AuNPs. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a biofunctional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of ~95 % in the presence of AuNPs. The 20 specificity of the ECL-modified tip was further studied by using more complex mixture of proteins which included non-glycosylated proteins and glycosylated proteins with different terminal sugar structure. Finally, the lectin affinity extraction device was tested with a real sample (E. coli cell lysate) spiked with target galactosylated glycoproteins.