Measurement of Neutral and Sialylated IgG N-Glycome at Asn-297 by CE-LIF to Assess Hypogalactosylation in Rheumatoid Arthritis.

Modulations in immunoglobulin G (IgG) N-glycosylation have been observed in many human diseases including chronic inflammatory diseases such as rheumatoid arthritis and also cancer. In this chapter, we describe how to determine hypogalactosylation for clinical samples, namely the sample preparation of IgG N-glycans at Asn-297 as well as the measurement of neutral and sialylated N-glycans by capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF).This semiautomated protocol describes the isolation polyclonal antibodies from serum, the separation of IgG-Fc glycopeptides from IgG antigen-binding fragment by pepsin digestion. Afterward, enzymatically released IgG-Fc N-glycans are cleaned up using a polyaromatic adsorbent resin followed by carbon purification. Sialic acids are then derivatized prior to glycan labeling. As a result, the agalactosylated N-glycan A2 does not co-migrate with sialylated N-glycans, which refines the measurement of hypogalactosylation by CE-LIF.