Exploring the scope of high throughput micro‐permethylation based glycomics.

Abstract

The glycosylation plays important roles in various molecular and cellular functions such as cell signaling, adhesion, development, immune recognition, lubrication, cellular defense etc. The protein glycosylation also contributes towards the stability, serum half‐life, immunogenicity and more importantly, biological activity of therapeutic glycoproteins, including antibodies, vaccines, biomarkers etc. This justifies the increasing demand for the qualitative and quantitative characterization and validation of glycosylation profile on glycoproteins. However, profiling of glycosylation is challenging owing to the glycosylation microheterogeneity, non‐template driven synthesis and their lower abundances. We have recently developed a high throughput N‐ and O‐glycomics strategy through a microscale permethylation based glycan derivatization followed by mass spectrometry (MS). Through this strategy facile but detailed quantitative structural profiling of glycans in a high throughput fashion can be attained. Here, we are reporting the wide applicability of our high throughput glycomics on glycoconjugates from various sources, including human serum, mammalian cells, milk oligosaccharides and purified glycoproteins via an automated tandem ESI‐MSn acquisition platform. The released or free glycans are permethylated in microscale in a 96‐well plate or microcentrifuge tubes and analyzed by an automated ESI‐MSn system after a C18 tip‐based cleanup. An internal standard based method was also incorporated for the absolute quantitation of glycan species. We have employed an in‐depth automated structural characterization MS program for N/O‐glycans through an automated ESI‐MSn acquisition up to MS5 level. Through this automated process, detailed glycan structural topology can be identified including glycan termini, branching, isomers, sialic acid linkages, and bisecting GlcNAc on the glycans. This approach can facilitate profiling of glycosylation on large sample sets, including clinical glycan biomarkers, milk oligosaccharides, and other glycoprotein therapeutics.Support or Funding InformationNational Institutes of Health grant (No. 1S10OD018530) to Dr. Parastoo Azadi