Sialic Acid Methylation Refines Capillary Electrophoresis Laser-Induced Fluorescence Analyses of Immunoglobulin G N-glycans of Ovarian Cancer Patients

Electrophoresis. 2014 Apr;35(7):1025-31. doi: 10.1002/elps.201300414.

Abstract

Alterations in IgG N-glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE-LIF of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans is a well-established rapid method to characterize IgG N-glycans that needs only low amounts of starting material. However,sialylated N-glycans have short migration times due to their negative charge. As a result,some of them are not well resolved and co-migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgGN-glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co-migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented,which allowed an accurate quantification of these N-glycans. Finally, we investigated the IgG N-glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods.With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Case-Control Studies
  • Electrophoresis, Capillary / methods*
  • Female
  • Humans
  • Immunoglobulin G / blood*
  • Immunoglobulin G / chemistry*
  • Methylation
  • N-Acetylneuraminic Acid / chemistry*
  • Ovarian Neoplasms / blood*
  • Ovarian Neoplasms / chemistry*
  • Polysaccharides / blood*
  • Polysaccharides / chemistry

Substances

  • Immunoglobulin G
  • Polysaccharides
  • glycosylated IgG
  • N-Acetylneuraminic Acid