Cellulose microspheres-filled pipet tips for purification and enrichment of glycans and glycopeptides

Abstract

The pretreatment of low-abundance glycans/glycopeptides prior to mass spectrometry (MS) analysis is of great significance for glycosylation identification of proteins. Herein, a simple one-step purification and enrichment procedure of glycans and glycopeptides was achieved using cellulose microspheres-filled pipet tips (CM-PTs). CM-PTs were fabricated by conveniently filling cellulose microspheres synthesized by sol-gel transition method into 100 μL pipet tips. The whole enrichment and purification process were completed within two minutes by pipetting up and down for dozens of times (30–40), followed by the direct analysis of MALDI TOF-MS. Upon the assessment of model glycoproteins, including human immunoglobulin G (IgG) and horse radish peroxidase (HRP), the CM-PT method exhibited good selectivity (IgG/BSA = 1:100), high sensitivity (1 ng μL−1 of IgG), large binding capacity (125 mg g−1, IgG/cellulose microspheres) and excellent reproducibility in glycopeptide enrichment. The capability for purifying glycans of CM-PTs was also investigated, presenting high binding capacity (8 mg g−1, maltoheptaose/cellulose microspheres) and good recoveries (81.3–89.8%). The developed in-pipet-tip system shows a great potential in efficient and high-throughput analysis of protein glycosylation.