Solid-Phase Chemical Modification for Sialic Acid Linkage Analysis: Application to Glycoproteins of Host Cells Used in Influenza Virus Propagation

Differentiation between the sialyl linkages is often critical to understanding biological consequence. Here we present a facile method for determining these linkages in glycans. Analysis of sialic acids is challenging due to their labile nature during sample preparation and ionization. Derivatization is often required via chemical reaction. Amidation derivatizes all sialic acids regardless of linkage, while esterification enables differentiation between alpha 2,3-linked and alpha 2,6-linked sialic acids. Reactions have been primarily performed on free glycans in solution but have been recently adapted to solid-phase providing unique advantages such as simplified sample preparation, improved yield, and high throughput applications. Here, we immobilized glycoproteins on resin-via reductive amination, modified alpha 2,6-linked sialic acids through ethyl esterification, and alpha 2,3-linked sialic acids via amidation. N-glycans and O-glycans were released via enzyme and chemical reactions. The method was applied for analysis of three different MDCK cell lines used for influenza propagation and where distributions of alpha 2,3 and alpha 2,6 sialic acids are critical for cell performance. Linkage specific distribution of these sialic acids was quantitatively determined and unique for each cell line. Our study demonstrates that protein sialylation can be reliably and quantitatively characterized in terms of sialic acid linkage of each glycan using the solid-phase esterification/amidation strategy.