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8 May / June 2017


Development of a Hydrophilic Interaction Liquid Chromatography Retention Model for Procainamide Tagged N-linked Glycans


by Emily Betchy, Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA; Barry Boyes, Advanced Materials Technology, Wilmington, DE, USA; Ron Orlando, Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA


A reproducible hydrophilic interaction liquid chromatography retention model has been developed for use with a fused-core Penta-HILIC column. This model is used for the identification of procainamide tagged N-linked glycans profiles based on their composition, with unique retentions for linkage and positional isomers. A dextran ladder reference was used to experimentally determine the retention of N-linked glycans from well characterised standards in glucose units, and coefficients representing the chromatographic influence of each glycan subunit were calculated via multi-variable linear regression. A model was developed using these coefficients, by which retention for individual glycans can be accurately predicted when utilising HILIC LCMS systems. This retention model provides an effective means of identifying isomeric glycoforms when employing HILIC LCMS analysis.


Introduction


Glycosylation is a form of co-translational and post-translational modification of proteins, involving an enzymatic process that results in the attachment of glycans to the proteins. N-linked glycans are attached to the nitrogen atom of an asparagine (Asn, N) residue that is part of an Asn-X-Ser/Thr consensus sequence (X can be any amino acid with the exception of proline) [1]. N-linked glycosylation is a dynamic process that allows cells to produce complex and diverse structures, which can be altered because of changes to the environment of the cell, making glycan research a challenging prospect [1]. The absolute prevalence of these glycosylated proteins in nature is not known, but protein databases currently suggest that over half of all proteins are N-glycosylated [2].


N-linked glycans fulfil several biological roles, both functional and structural, from cell signalling and interaction to protein folding and immune responses [1,3]. N-linked glycans on glycoproteins have been the subject of many epidemiological research studies, resulting in greater understanding of various diseases such as various cancers and influenza [1,4-15]. Additionally, changes in the abundance and


Figure 1: Visual representation of the correlation between the dextran ladder glucose units and the N-linked glycans.


structure of glycans have been and continue to be explored as biomarkers for different disease states [10,13-15]. The recent advances in research have served to raise awareness that the ability to ascertain more refined linkage and position information is of vital importance, as structure is proving


to be of critical importance to the function of a glycan. Even the smallest of changes in linkage can produce a significant change in function, therefore it is essential that analytical methods be able to reliably characterise glycans.


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