Abstract
Glycosylation is one of the most common and complex post-translational modifications of proteins. However, there are other carbohydrates such as free oligosaccharides and glycosphingolipids-glycans that are associated with important biological and clinical roles. To analyze these molecules using liquid chromatography coupled with mass spectrometry (LC-MS), the permethylation approach was utilized. Although permethylation is a commonly utilized glycan derivatization technique, separation of permethylated glycans released from glycosphingolipid (GSL) by LC-MS has never been previously demonstrated. Here, a nanoflow porous graphitized carbon (PGC) column coupled with a high-resolution mass spectrometer was used to achieve isomeric separation of these permethylated glycans. We demonstrate the separation of free reducing end and reduced end O-glycans, free oligosaccharides derived from human milk, and GSL glycans derived from the MDA-MB-231BR cancer cell line using PGC-LC-MS.
Highlights
As a post-translational modification (PTM), glycosylation is one of the most complex and perhaps the most essential modifications of the protein [1]
To examine the viability of O-glycan separation, O-glycans derived from standard glycoproteins were first assessed
We demonstrated isomeric separation of permethylated O-glycans, free oligosaccharides, and GSL-glycans using a porous graphitized carbon (PGC)-LC technique that has been previously shown to separate permethylated N-glycans [28,29,30,31]
Summary
As a post-translational modification (PTM), glycosylation is one of the most complex and perhaps the most essential modifications of the protein [1]. Due to the many important physiological roles that glycans play, their importance has been frequently highlighted by researchers from multiple disciplines. Glycans have been associated with coronaviruses’ spike glycoproteins, including that SARS-CoV-2 virion that causes COVID-19 [17]. Glycosylation is most frequently associated with protein glycosylation, there are multifarious glycoconjugates, including glycolipids [18,19]. N-glycosylation and O-glycosylation of proteins are the two major glycoconjugates. N-glycosylation involves glycans with a specific core structure being attached to the asparagine, with asparagine-X-serine/threonine amino acid sequence and X being any amino acid (except proline). O-glycosylation, on the other hand, occurs on the serine or threonine residue with no specific core structure, yielding a more complex structure [20]
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