Abstract
In affinity chromatography, enrichment of biomolecules is dependent on the selection of affinity sites immobilized onto a suitable support material. A few hydrazide - functionalized materials with surface modification protocols compatible to conventional support materials like silica and cellulose are reported. The study demonstrates the modification/derivatization pathways that can be adopted to modify the support materials with similar surface chemistry like cellulose, poly(GMA/DVB), or diamond. Poly(GMA/DVB) and cellulose represent hydrophilic supports whereas diamond is a hydrophobic support material. SEM images of three materials provide surface morphology whereas FT-IR confirms reaction completion and derivatization. These hydrazide - functionalized materials are applied to fetuin digest for glycopeptides enrichment and subsequently for selectivity and sensitivity assessment. Statistically, poly(GMA/DVB) shows 85.7% sensitivity with specificity of 88.8% in the enrichment experiments. Diamond offers hydrophobic interactions to non-glycopeptides and they co-elute with glycopeptides, resulting in reduced sensitivity down to 69.2%. Poly(GMA/DVB) shows recovery up to 89%, while recovery for cellulose and diamond is 83 and 71%, respectively. The materials enrich mono-N-linked-glycosylated peptide from tryptic digest of chicken avidin spiked in fetuin digest. The hydrazide group density on cellulose, poly(GMA/DVB), and diamond is 2.8, 2.3, and 2.1 mmol/g, respectively; this contributes towards the specificity and sensitivity of designed materials. The materials are also applied to serum samples and enriched glycopeptides characteristic of serum glycoproteins of clinical importance. Therefore this study provides routes for the economical surface modifications of support materials and to fabricate affinity materials with improved efficiency.
Graphical Abstract
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