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Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan-Relative Quantification Strategy

  • Research Article
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Journal of The American Society for Mass Spectrometry

Abstract

The Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT) strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction attributable to isotopic overlap and a post-acquisition normalization of the data to account for any systematic bias, a plot of the experimental H:L ratio versus the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics.

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Acknowledgments

The authors gratefully acknowledge financial support received from the NIH – NCI IMAT Program (grant #R33 CA147988-02), the W. M. Keck Foundation, and North Carolina State University.

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  1. Department of Chemistry, W. M. Keck Fourier Transform Mass Spectrometry Laboratory, North Carolina State University, Raleigh, NC, 27695, USA

    S. Hunter Walker, Amber D. Taylor & David C. Muddiman

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  1. S. Hunter Walker
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  3. David C. Muddiman
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Correspondence to David C. Muddiman.

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Walker, S.H., Taylor, A.D. & Muddiman, D.C. Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan-Relative Quantification Strategy. J. Am. Soc. Mass Spectrom. 24, 1376–1384 (2013). https://doi.org/10.1007/s13361-013-0681-2

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