Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Glycobiology
Jeffrey S RohrerNebojsa Avdalovic

Abstract

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 microm recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was...Continue Reading

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Citations

Dec 3, 2013·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Dan WangXue-Long Sun
Nov 15, 2001·FEBS Letters·S BürgmayrE Staudacher
Dec 15, 2005·Journal of the American Society for Mass Spectrometry·James J WaltersJames Bashkin
Nov 26, 2015·MAbs·Lei ZhangBaolin Zhang
May 10, 2011·Biologicals : Journal of the International Association of Biological Standardization·Akira HarazonoTeruhide Yamaguchi
Oct 18, 2006·Molecular Reproduction and Development·José Guillermo VelásquezPilar Coy
Sep 4, 2004·Journal of Biochemical and Biophysical Methods·Michael WeitzhandlerNebojsa Avdalovic
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Dec 18, 2014·Nature Communications·Preston S K NgMichael P Jennings
Oct 19, 2016·Accounts of Chemical Research·Manveen K SethiSusan Fanayan
Apr 4, 2017·Electrophoresis·Lucas VeillonYehia Mechref
Aug 6, 2013·Molecular & Cellular Proteomics : MCP·Elodie Mathieu-RivetMichael Hippler
Dec 23, 2017·Reproduction : the Official Journal of the Society for the Study of Fertility·Yuanxian WangWuzi Dong
Sep 12, 2020·Journal of Separation Science·Bastiaan L DuivelshofValentina D'Atri
Oct 14, 2017·Analytical and Bioanalytical Chemistry·Matthias MechelkePetra Kornberger
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Apr 11, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Zhi-P CaiJosef Voglmeir

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