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

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.