Novel Strategy to Release and Tag N-Glycans for Functional Glycomics

Abstract

Functional glycomics has been impeded by the lack of inexpensive enzymatic and mild chemical methods to acquire natural glycans in significant amounts. In this study, we have developed a new strategy we term “threshing and trimming” (TaT) to quickly obtain N-glycans from glycoproteins and animal tissues. TaT employs low-cost Pronase to degrade peptides and N-bromosuccinimide (NBS) to effect oxidative decarboxylation under very mild reaction conditions to generate homogeneous aglycon moieties as nitriles or aldehydes. These aglycons can be readily conjugated with fluorescent tags for profiling and functional study. TaT is an affordable alternative to expensive specialty enzymes and strong chemical treatment and unpleasant reagents, and should further drive the functional glycomics of N-glycans.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…Work in this area suffers from the lack of inexpensive enzymatic and mild chemical methods to obtain N ‐glycans in significant amounts for some structural (e.g., NMR) and biochemical studies. To address this problem, Song et al () have developed a method which they term “threshing and trimming” (TaT) to obtain N ‐glycans from glycoproteins or animal tissues. Pronase was used to hydrolyze the peptide chain and N ‐bromosuccinimide (NBS) was then used to produce glycans as nitriles by oxidative decarboxylation.…”

Preparation of Complex Glycans From Natural Sources for Functional Study

“…Recently, we reported two different chemical approaches for large-scale release of N-glycans. The first approach is a “chemoenzymatic” method to release N-glycans called threshing and trimming (TaT) (Song et al, 2014b ). In the first threshing step, glycoproteins are treated with pronase to create a pool of N-glycoamino acids and glycopeptides with short peptide moieties.…”

Natural and Synthetic Sialylated Glycan Microarrays and Their Applications

“…These arrays are often presented as shotgun arrays, meaning that the glycans are not sequenced prior to printing and as such, the investigators are reliant on interactions with GBPs to prioritize the glycan characterization. Multiple strategies for glycan release from the tissue have been outlined including enzymatic release (Heimburg-Molinaro et al, 2011; Song et al, 2014) and chemical release using sodium hypochlorite (NaOCl or bleach) (Song et al, 2016). Once the glycans have been released, the functionalization process varies widely with respect to the linkers used, the substrate for printing, and the methods of analysis.…”