Glycans are post-translational modifications on proteins that play a significant role in protein activity. N-glycans are linked to Asparagine, and O-glycans are linked to either Serine or Threonine residue; the structural features of these glycans are crucial for their activity. Decades of research have been devoted to the characterization and modification of glycans that occur during disease progression. Research has shown N and O-glycan modification to be a hallmark for various cancers; the focus needs to be given to accurate release methods and quantification. With the advent of mass spectrometry, the study and characterisation of glycans have been revolutionised [1]. Peptide: N-glycosidase F (PNGase F) is an enzyme highly efficacious and specially tailored for N-glycans, contrary to that O-glycans are released using chemical reductive beta-elimination method, causing the loss of important modifications such as O-acetylation. O-acetylation is a common modification of sialic acids that has been implicated in a multitude of biological and disease processes. A lack of analytical methods that can determine these modifications is essential to identify their true biological functions.
Recently Vos et al, demonstrated the oxidative release of O-Glycans under neutral conditions for glycans with base-sensitive substituents such as acetylation [2]. We further this work by optimising the oxidative release of O-glycans from glycoproteins immobilised on PVDF. Additionally, we are able to demonstrate the separation of isomeric O-acetylation sialosides on O-glycans using porous graphitised carbon (PGC) separation and negative mode tandem mass spectrometry.
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