GlcNAc, chemically known as N-acetyl-2-amino-2-deoxy-D-glucose, is a glucose- and glucosamine-bound in vivo precursor of downstream products of glucose metabolism. GlcNAc is a key component of bacterial and fungal cell walls and the extracellular matrix of animal cells. It plays a variety of roles in cell surface structure and is under discussion to be involved in signaling pathways. GlcNAc alters multiple functions of proteins involved in a variety of biological processes. Glycosylation modifications are involved in the pathophysiological processes of a variety of diseases such as diabetes, Alzheimer's disease, and heart disease, so it is of positive significance to study GlcNAc.
Fig.1 Protein N-glycosylation reaction. (de-Souza-Ferreira, et al., 2023)
Separation of naturally occurring isomers with the same mass and similar fragmentation patterns is a prerequisite for their accurate quantification but remains a challenging task in Carbohydrate Metabolism Analysis. The low concentration of many isomers (i.e., sugars and their derivatives) in complex and concentrated matrices requires efficient separation methods that tolerate high sample loads, as well as selective and sensitive detection methods. CD BioGlyco uses a method based on the highly efficient separation of ethoxylated and trimethylsilylated GlcNAc using a biologically derived. We use the 13C-labeled internal standard method, which allows for accurate absolute quantification and eliminates systematic errors associated with sample pretreatment and analysis. Gas chromatography (GC) provides high sample capacity, separation efficiency, and precision under measured reproducibility conditions, which are necessary for analyzing low-abundance stereoisomers in biological samples.
Fig.2 Flowchart of GlcNAc analysis. (CD BioGlyco)
Paper Title: Selective and accurate quantification of N-acetylglucosamine in biotechnological cell samples via GC-MS/MS and GC-TOFMS
Technology: GC-MS/MS, GC-TOFMS
Journal: Analytica Chemistry
Results: A two-step derivatization was used to convert the polar functional groups to ensure their suitability for gas chromatography. In the first step, alkoxylation converts the aldehyde carbon group of N-acetyl hexosamine to an alkoxy amino group, forming cis and trans isomers by opening the ring structure. In the second step, trimethylsilylation replaces the active hydrogen atom of the hydroxyl group. For all combinations tested, the best separations were obtained for the ethoxylated derivatives on a 100% dimethylpolysiloxane column, as shown in Figure 3 (baseline separation of ManNAc and GlcNAc within 16 min). In this work, a robust and highly selective GC method for the detection of GlcNAc separation was validated using a 5% phenyl 95% dimethylpolysiloxane column (60 m × 0.25 mm × 0.25 μm) and successfully used for the analysis of biotechnology-related Penicillium chrysogenum samples.
Fig.3 Extracted ion chromatograms (GC−CI-TOF) of the three biologically relevant N-acetylated hexosamines. (Mairinger, et al., 2020)
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