Glycans significantly influence the therapeutic efficacy of glycoprotein by regulating its stability, serum half-life, immunogenicity and biological activity. The demand for the qualitative and quantitative characterization and validation of glycosylation on therapeutic glycoproteins which includes antibodies, vaccines, biomarkers etc. are increasing rapidly. One of the classical yet most informative derivatization methods for glycan characterization is the permethylation of glycans released from glycoproteins and subsequent characterization by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Electrospray Ionization (ESI-MS). Even though manual permethylation and subsequent processing of glycans is still highly prevalent, all reactions are still performed manually, on one sample at a time. We have developed a cost-effective, simple, sensitive and rapid permethylation kit for the micro-permethylation of glycans released from glycoproteins.
Here, we report a protocol for the automation of permethylation reaction in microscale that allows processing of samples in a small amount in a high throughput fashion with minimal handling. The protocol involves releasing of N/O-glycans from glycoprotein samples, the permethylation reaction in microscale, extraction of permethylated glycans directly in a 96 well plate or microcentrifuge tube with significantly shorter processing time and efforts. The permethylated glycans are subsequently analyzed by mass spectrometry. The workflow can be applied to purified glycoproteins or mixtures of glycans or glycopeptides. Automation of permethylation enables high throughput screening of glycosylation for glycomics on a large scale for clinical and biomarker studies which allows for convenient, faster, and reliable characterization of glycoforms.