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Demonstration of hydrazide tagging for O-glycans and a central composite design of experiments optimization using the INLIGHT™ reagent

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Abstract

The INLIGHT™ strategy for N-linked glycan derivatization has been shown to overcome many of the challenges associated with glycan analysis. The hydrazide tag reacts efficiently with the glycans, increasing their non-polar surface area, allowing for reversed-phase separations and increased ionization efficiency. We have taken the INLIGHT™ strategy and adopted it for use with O-linked glycans. A central composite design was utilized to find optimized tagging conditions (45% acetic acid, 0.1 μg/μL tag concentration, 37 C, 1.75 h). Derivatization at optimized conditions was much quicker than any hydrazide derivatization strategy used previously. Human immunoglobulin A (IgA) and bovine submaxillary mucin (BSM) were then deglycosylated through hydrazinolysis and the removed glycans were tagged under optimum conditions. XIC of tagged glycans and MS2 data show successful hydrazide tagging of O-linked glycans for the first time.

The INLIGHT™ hydrazide tag was optimized using a central composite design for derivatization of O-linked glycans. Two glycoprotein standards were deglycosylated through hydrazinolysis and tagged at the optimized conditions. MS/MS data shows INLIGHT™ derivatization of glycans demonstrating successful hydrazide tagging of O-glycans for the first time.

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Acknowledgements

This work was supported by the North Carolina State University Chemistry Graduate Assistantship and the American Chemical Society Division of Analytical Chemistry Fellowship, sponsored by Eli Lilly Inc. The Hydrazinolysis protocol was graciously provided thanks to Dr. Parastoo Azadi at the Complex Carbohydrates Research Center.

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Authors and Affiliations

  1. W. M. Keck FTMS Laboratory from Human Health Research, Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA

    Samuel R. King, Elizabeth S. Hecht & David C. Muddiman

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  1. Samuel R. King
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  2. Elizabeth S. Hecht
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  3. David C. Muddiman
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Correspondence to David C. Muddiman.

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King, S.R., Hecht, E.S. & Muddiman, D.C. Demonstration of hydrazide tagging for O-glycans and a central composite design of experiments optimization using the INLIGHT™ reagent. Anal Bioanal Chem 410, 1409–1415 (2018). https://doi.org/10.1007/s00216-017-0828-2

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  • DOI: https://doi.org/10.1007/s00216-017-0828-2

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