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.