Glycosylation heterogeneity and low abundant serum glycoproteins MS analysis by boronic acid immobilized Fe3O4@1,2-Epoxy-5-Hexene/DVB magnetic core shell nanoparticles

Abstract

Glycosylation in biological systems has been studied by enriching glyco- moieties followed by their mass spectrometric (MS) analyses. Complexity of glycoproteins heterogeneity is addressed in this study. Mono- and multi-glycosylated peptides after proteolysis from complex biological samples are enriched by newly synthesized boronic acid functionalized magnetic 1,2-epoxy-5-hexene/DVB core shell nanoparticles under optimized pH conditions. The composition, surface area, structure and morphology are found out by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and nitrogen adsorption porosimetry. Glycopeptides enrichment efficiency is assessed by using tryptic digests of Horseradish Protease with 8 and Immunoglobulin G with single glycosylation site, identifying 17 and 26 glycopeptides respectively by MALDI-mass spectrometry representing complete glycosylation sites coverage. The material shows low sensitivity (0.1 fmol µL−1), high selectivity (1:250) using bovine serum albumin digest, high adsorption capacity (332 mg g−1 of horseradish peroxidase) and reusability (up to three times), providing cost-effective in-house solution to study complex proteolytic samples. The application of magnetic polymeric core shell NPs to digested human serum helps identifying 347 glycopeptides by nano-LC-MS/MS, which also include less abundant glycoproteins like ceruloplasmin, complement C4 and acid alpha-1-glycoprotein. The ease of sample preparation because of magnetic nature, high selectivity and sensitivity promise a tool to study glycoproteins of clinical value.