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Global site-specific analysis of glycoprotein N-glycan processing

Nature Protocols volume 13, pages 1196–1212 (2018)Cite this article

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Abstract

N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high-mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge, we developed a robust semiquantitative mass spectrometry (MS)-based method that determines the degree of glycan occupancy at each glycosite and the proportion of N-glycans processed from high-mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 μg of protein. Here, we provide a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (iv) LC-MS/MS analysis; and (v) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved, with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/postdoc with basic skills for proteomics experiments and takes ∼7 d to complete.

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Figure 1: N-linked glycan processing in the endoplasmic reticulum and Golgi apparatus.
Figure 2: Schematic overview of the protocol.
Figure 3: Validation of sequential endoglycosidase treatment.
Figure 4: Validation of MS detection of glycotypes.
Figure 5: Application of the protocol for characterization of site-specific N-glycan processing of recombinant glycoprotein therapeutics and serum glycoproteins.
Figure 6: Application of the protocol for characterization of site-specific N-glycan processing of virus envelope glycoproteins.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grants R01AI113867 (J.C.P., J.R.Y. and W.R. Schief), UM1 AI100663 (D.R.B. and J.C.P.), R01AI127521 (J.S.M.), and P41 GM103533 (J.R.Y.).

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Authors and Affiliations

  1. Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA

    Liwei Cao, Jolene K Diedrich, Yuanhui Ma, Sung-Kyu Robin Park, Claire M Delahunty, John R Yates & James C Paulson

  2. Department of Immunology and Microbiology, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California, USA

    Liwei Cao, Matthias Pauthner, Dennis R Burton & James C Paulson

  3. Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA

    Nianshuang Wang & Jason S McLellan

  4. Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA

    Dennis R Burton

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Contributions

L.C., J.R.Y., and J.C.P. designed the research. L.C. prepared samples for MS analysis. J.K.D., L.C., C.M.D., and Y.M. performed the MS analysis. L.C. and S.-K.R.P. analyzed the data. N.W. and M.P. expressed and purified Env proteins. J.S.M., D.R.B., J.R.Y., and J.C.P. supervised the project. L.C. and J.C.P. wrote the manuscript.

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Correspondence to James C Paulson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of peptides with multiple glycosylation sites with the protocol.

MS/MS spectra and fragment assignment of the (a) di-glycosylated, (b) tri-glycosylated, and (c) tetra-glycosylated peptides derived from BG505 SOSIP.664.

Supplementary Figure 2 Scatter plot of site-specific N-glycan processing of MERS-CoV S protein with a breakdown on the used proteases.

(a) Triple digestion, (b) the combination of trypsin and chymotrypsin, (c) chymotrypsin. The proportions of high mannose and complex type glycans at those glycosites highlighted in yellow were assigned based on the proportion of spectra hits since peak area did not reach the threshold. Mean ± SEM were plotted.

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Supplementary Figures 1 and 2, and Supplementary Tables 1–5. (PDF 503 kb)

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Cao, L., Diedrich, J., Ma, Y. et al. Global site-specific analysis of glycoprotein N-glycan processing. Nat Protoc 13, 1196–1212 (2018). https://doi.org/10.1038/nprot.2018.024

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