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Simultaneous quantification of N- and O-glycans using a solid-phase method

Nature Protocols volume 12, pages 1229–1244 (2017)Cite this article

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Abstract

Glycosylation has a pivotal role in a diverse range of biological activities, modulating the structure and function of proteins. Glycogens coupled to the nitrogen atom (N-linked) of asparagine side chains or to the oxygen atom (O-linked) of serine and threonine side chains represent the two major protein glycosylation forms. N-glycans can be released by glycosidases, whereas O-glycans are often cleaved by chemical reaction. However, it is challenging to combine these enzymatic and chemical reactions in order to analyze both N- and O-glycans. We recently developed a glycoprotei n immobilization for glycan extraction (GIG) method that allows for the simultaneous analysis of N- and O-glycans on a solid support. GIG enables quantitative analysis of N-glycans and O-glycans from a single specimen and can be applied to a high-throughput automated platform. Here we provide a step-by-step GIG protocol that includes procedures for (i) protein immobilization on an aldehyde-active solid support by reductive amination; (ii) stabilization of fragile sialic acids by carbodiimide coupling; (iii) release of N-glycans by PNGase F digestion; (iv) release of O-glycans by β-elimination using ammonia in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP) to prevent alditol peeling from O-glycans; (v) mass spectrometry (MS) analysis; and (vi) data analysis for identification of glycans using in-house developed software (GIG Tool; free to download via http://www.biomarkercenter.org/gigtool). The GIG tool extracts precursor masses, oxonium ions and glycan fragments from tandem (liquid chromatography (LC)–MS/MS) mass spectra for glycan identification, and reporter ions from quaternary amine containing isobaric tag for glycan (QUANTITY) isobaric tags are used for quantification of the relative abundance of N-glycans. The GIG protocol takes ∼3 d.

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Figure 1: Schematic diagram of sequential release of N-linked and O-linked glycans via treatment.
Figure 2: O-glycan profiling of mucin from porcine stomach (MPS) by ESI–MS.
Figure 3: Chemoenzymatic sequential release of N-glycans and O-glycans from bovine-serum-derived fetuin using GIG.
Figure 4: Sialylated O-glycans of mucin from bovine submaxillary glands by MALDI–MS.

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Acknowledgements

We thank T. Stefani and P. Shah from Johns Hopkins University for help with LC–MS and Shimadzu for providing the instrument for MALDI–MS. This work was supported by the National Institutes of Health, National Cancer Institute, the Early Detection Research Network (EDRN; U01CA152813), the Clinical Proteomic Tumor Analysis Consortium (CPTAC; U24CA160036) and the National Institutes of Health, National Heart Lung and Blood Institute Programs of Excellence in Glycosciences (P01HL107153) and the Johns Hopkins Proteomics Center (N01-HV-00240).

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  1. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Shuang Yang, Yingwei Hu, Lori Sokoll & Hui Zhang

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  1. Shuang Yang
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Contributions

S.Y. and H.Z. designed the research. H.Z. and L.S. directed the project. S.Y. developed the experimental protocol and conducted the experiments. S.Y. wrote the manuscript. Y.H. developed the data analysis tools and provided support with the data analysis.

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Correspondence to Shuang Yang or Hui Zhang.

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The authors declare no competing financial interests.

Supplementary information

Combo PDF

The Supplementary Note and Supplementary Table 1. (PDF 340 kb)

Supplementary Software 1

Format template for N-glycan database file (nlib.csv). (ZIP 164 kb)

Supplementary Software 2

Format template for O-glycan database file (olib.csv). (ZIP 424 kb)

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Yang, S., Hu, Y., Sokoll, L. et al. Simultaneous quantification of N- and O-glycans using a solid-phase method. Nat Protoc 12, 1229–1244 (2017). https://doi.org/10.1038/nprot.2017.034

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