µSI Process Analyzer System for Glycan Map Sample Prep
In this implementation, N-linked oligosaccharides are enzymatically released from samples using Peptide-N-Glycosidase F (PNGase F). The free oligosaccharides are then derivatized with the fluorophore 2-aminobenzoic acid (2-AA) on the reducing terminal N-acetylglucosamine (GlcNAc). The fluorescently labeled oligosaccharides are separated by the HILIC analytical column and detected by fluorescence. InFigure 2 , the µSI process analyzer system replaces manual processes with automated sampling, sample purification and matrix removal, enzymatic release of glycans, fluorescent labeling of glycans, enrichment of labeled glycans, and synchronized injection, along with chromatographic separation and quantitation of labeled glycans.
Table 2 describes the glycan map sample preparation with protein capture, sample matrix removal, and glycan release by on-column enzymatic digestion in the µSI process analyzer system. InFigure 2a , cell-free cell culture samples are acquired by the aspiration action of syringe pump 1 and sent to the affinity purification column, where the protein of interest is retained in the column, and the sample matrix is removed as column flow-through. The enzyme is subsequently introduced into the affinity column, and glycans are released in the interstitial solution of the affinity column. This design eliminates the need for protein precipitation. The released glycans are carried out by syringe pump 1 and mixed with the glycan reagent from syringe pump 2 before the degasser. The degasser removes microbubbles formed when the aqueous solution from the affinity column meets the organic solution of the 2-AA reagent prepared in methanol. The degassed glycans and 2-AA reagent mixture are delivered to the 80°C reaction coil to accelerate the fluorescent labeling reaction in flow-through mode. The 2-AA labeled glycans are air-cooled and enriched by a porous graphitic carbon (PGC) column while the excess 2-AA reagent and non-labeled glycans are cleared as the PGC column flow-through. The enriched labeled glycans are released from the PGC column and transferred to an LC injection valve, where glycan quantitation is accomplished through HILIC chromatography.