Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Varki, A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130 (1993).

    CAS  PubMed  Article  Google Scholar 

  2. 2

    Kellokumpu, S., Sormunen, R. & Kellokumpu, I. Abnormal glycosylation and altered Golgi structure in colorectal cancer: dependence on intra-Golgi pH. FEBS Lett. 516, 217–224 (2002).

    CAS  PubMed  Article  Google Scholar 

  3. 3

    Dube, D.H. & Bertozzi, C.R. Glycans in cancer and inflammation—potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4, 477–488 (2005).

    CAS  PubMed  Article  Google Scholar 

  4. 4

    Wang, J.-Z., Grundke-Iqbal, I. & Iqbal, K. Glycosylation of microtubule-associated protein tau: An abnormal posttranslational modification in Alzheimer′s disease. Nat. Med. 2, 871–875 (1996).

    CAS  Article  Google Scholar 

  5. 5

    Itoh, N. et al. Analysis of N-glycan in serum glycoproteins from db/db mice and humans with type 2 diabetes. Am. J. Physiol. Endocrinol. Metab. 293, E1069–E1077 (2007).

    CAS  PubMed  Article  Google Scholar 

  6. 6

    Montpetit, M.L. et al. Regulated and aberrant glycosylation modulate cardiac electrical signaling. Proc. Natl. Acad. Sci. USA 106, 16517–16522 (2009).

    CAS  PubMed  Article  Google Scholar 

  7. 7

    Yang, S. et al. Glycoproteins identified from heart failure and treatment models. Proteomics 15, 567–579 (2015).

    CAS  PubMed  Article  Google Scholar 

  8. 8

    Wang, X. et al. Overexpression of α (1, 6) fucosyltransferase associated with aggressive prostate cancer. Glycobiology 24, 935–944 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9

    Peracaula, R., Barrabés, S., Sarrats, A., Rudd, P.M. & de Llorens, R. Altered glycosylation in tumours focused to cancer diagnosis. Dis. Markers 25, 207–218 (2008).

    CAS  PubMed  Article  Google Scholar 

  10. 10

    Hakomori, S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc. Natl. Acad. Sci. USA 99, 10231–10233 (2002).

    CAS  PubMed  Article  Google Scholar 

  11. 11

    Hamid, U.M.A. et al. A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression. Glycobiology 18, 1105–1118 (2008).

    Article  CAS  Google Scholar 

  12. 12

    Iyer, R. & Carlson, D.M. Alkaline borohydride degradation of blood group H substance. Arch. Biochem. Biophys. 142, 101–105 (1971).

    CAS  PubMed  Article  Google Scholar 

  13. 13

    Cummings, R. et al. Biosynthesis of N-and O-linked oligosaccharides of the low density lipoprotein receptor. J. Biol. Chem. 258, 15261–15273 (1983).

    CAS  PubMed  Google Scholar 

  14. 14

    Takasaki, S., Mizuochi, T. & Kobata, A. Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides. Methods Enzymol. 83, 263–268 (1981).

    Article  Google Scholar 

  15. 15

    Patel, T. et al. Use of hydrazine to release in intact and unreduced form both N-and O-linked oligosaccharides from glycoproteins. Biochemistry 32, 679–693 (1993).

    CAS  PubMed  Article  Google Scholar 

  16. 16

    Merry, A.H. et al. Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis. Anal. Biochem. 304, 91–99 (2002).

    CAS  PubMed  Article  Google Scholar 

  17. 17

    Zauner, G., Koeleman, C.A., Deelder, A.M. & Wuhrer, M. Mass spectrometric O-glycan analysis after combined O-glycan release by beta-elimination and 1-phenyl-3-methyl-5-pyrazolone labeling. Biochem. Biophys. Acta 1820, 1420–1428 (2012).

    CAS  PubMed  Article  Google Scholar 

  18. 18

    Huang, Y., Mechref, Y. & Novotny, M.V. Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis. Anal. Chem. 73, 6063–6069 (2001).

    CAS  PubMed  Article  Google Scholar 

  19. 19

    Honda, S. et al. High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives. Anal. Biochem. 180, 351–357 (1989).

    CAS  PubMed  Article  Google Scholar 

  20. 20

    Yang, S., Li, Y., Shah, P. & Zhang, H. Glycomic analysis using glycoprotein immobilization for glycan extraction. Anal. Chem. 85, 5555–5561 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21

    Yang, S. & Zhang, H. Glycomic analysis of glycans released from glycoproteins using chemical immobilization and mass spectrometry. Curr. Protoc. Chem. Biol. 6, 191–208 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  22. 22

    Sekiya, S., Wada, Y. & Tanaka, K. Derivatization for stabilizing sialic acids in MALDI-MS. Anal. Chem. 77, 4962–4968 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23

    Weiskopf, A.S., Vouros, P. & Harvey, D.J. Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides. Anal. Chem. 70, 4441–4447 (1998).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24

    Yang, S. & Zhang, H. Glycan analysis by reversible reaction to hydrazide beads and mass spectrometry. Anal. Chem. 84, 2232–2238 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25

    Shah, P. et al. Mass spectrometric analysis of sialylated glycans with use of solid-phase labeling of sialic acids. Anal. Chem. 85, 3606–3613 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26

    Powell, A.K. & Harvey, D.J. Stabilization of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 10, 1027–1032 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27

    Reiding, K.R., Blank, D., Kuijper, D.M., Deelder, A.M. & Wuhrer, M. High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification. Anal. Chem. 86, 5784–5793 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28

    Chen, P., Werner-Zwanziger, U., Wiesler, D., Pagel, M. & Novotny, M.V. Mass spectrometric analysis of benzoylated sialooligosaccharides and differentiation of terminal α2→ 3 and α2→ 6 sialogalactosylated linkages at subpicomole levels. Anal. Chem. 71, 4969–4973 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29

    North, S.J., Hitchen, P.G., Haslam, S.M. & Dell, A. Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr. Opin. Struct. Biol. 19, 498–506 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30

    Mariño, K., Bones, J., Kattla, J.J. & Rudd, P.M. A systematic approach to protein glycosylation analysis: a path through the maze. Nat. Chem. Biol. 6, 713–723 (2010).

    PubMed  Article  CAS  Google Scholar 

  31. 31

    Yang, S., Rubin, A., Eshghi, S.T. & Zhang, H. Chemoenzymatic method for glycomics: isolation, identification, and quantitation. Proteomics 16, 241–256 (2016).

    CAS  PubMed  Article  Google Scholar 

  32. 32

    Reinhold, V., Zhang, H., Hanneman, A. & Ashline, D. Toward a platform for comprehensive glycan sequencing. Mol. Cell Proteomics 12, 866–873 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33

    Royle, L. et al. HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal. Biochem. 376, 1–12 (2008).

    CAS  PubMed  Article  Google Scholar 

  34. 34

    Young, N.M. et al. Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277, 42530–42539 (2002).

    CAS  PubMed  Article  Google Scholar 

  35. 35

    Lauc, G. & Wuhrer, M. High-Throughput Glycomics and Glycoproteomics: Methods and Protocols (Methods in Molecular Biology) (Humana Press, 2017).

  36. 36

    Stavenhagen, K., Plomp, R. & Wuhrer, M. Site-specific protein N-and O-glycosylation analysis by a C18-porous graphitized carbon–liquid chromatography-electrospray ionization mass spectrometry approach using pronase treated glycopeptides. Anal. Chem. 87, 11691–11699 (2015).

    CAS  PubMed  Article  Google Scholar 

  37. 37

    Yang, S. et al. QUANTITY: an isobaric tag for quantitative glycomics. Sci. Rep. 5, 17585 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Furukawa, J et al. A versatile method for analysis of serine/threonine posttranslational modifications by β-elimination in the presence of pyrazolone analogues. Anal. Chem. 83, 9060–9067 (2011).

    CAS  PubMed  Article  Google Scholar 

  39. 39

    Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J.A. & Prior, R.L. High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J. Agric. Food Chem. 50, 4437–4444 (2002).

    CAS  PubMed  Article  Google Scholar 

  40. 40

    Chen, J., Shah, P. & Zhang, H. Solid phase extraction of N-linked glycopeptides using hydrazide tip. Anal. Chem. 85, 10670–10674 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41

    Mach, A.J., Kim, J.H., Arshi, A., Hur, S.C. & Di Carlo, D. Automated cellular sample preparation using a centrifuge-on-a-chip. Lab Chip 11, 2827–2834 (2011).

    CAS  PubMed  Article  Google Scholar 

  42. 42

    Zhou, H., Ranish, J.A., Watts, J.D. & Aebersold, R. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat. Biotechnol. 20, 512–515 (2002).

    CAS  PubMed  Article  Google Scholar 

  43. 43

    Stöckmann, H., Adamczyk, B., Hayes, J. & Rudd, P.M. Automated, high-throughput IgG-antibody glycoprofiling platform. Anal. Chem. 85, 8841–8849 (2013).

    PubMed  Article  CAS  Google Scholar 

  44. 44

    Shah, P. et al. Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveal glycoprotein alteration in protein abundance and glycosylation. Mol. Cell Proteomics 14, 2753–2763 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45

    Sun, S. et al. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nat. Biotechnol. 34, 84–88 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46

    Yang, S. et al. Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy. Anal. Chem. 87, 9671–9678 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47

    Yin, B. et al. Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation. Biotechnol. Bioeng. 112, 2343–2351 (2015).

    CAS  PubMed  Article  Google Scholar 

  48. 48

    Yang, W. et al. Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy. Anal. Chem. 86, 6959–6967 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49

    Chung, C.Y. et al. Integrated genome and protein editing swaps α-2, 6 sialylation for α-2, 3 sialic acid on recombinant antibodies from CHO. Biotechnol. J. 12, 1600502 (2016).

    Article  CAS  Google Scholar 

  50. 50

    Do, D.C. et al. N-glycan in cockroach allergen regulates human basophil function. J. Allergy Clin. Immunol. 139, AB167 (2017).

    Article  Google Scholar 

  51. 51

    Yang, S. et al. Glycan analysis by isobaric aldehyde reactive tags and mass spectrometry. Anal. Chem. 85, 8188–8195 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52

    Lauber, M.A. et al. Rapid preparation of released N-glycans for HILIC analysis using a labeling reagent that facilitates sensitive fluorescence and ESI-MS detection. Anal. Chem. 87, 5401–5409 (2015).

    CAS  PubMed  Article  Google Scholar 

  53. 53

    Harvey, D.J. Proteomic analysis of glycosylation: structural determination of N-and O-linked glycans by mass spectrometry. Expert Rev. Proteomics 2, 87–101 (2005).

    CAS  PubMed  Article  Google Scholar 

  54. 54

    Brockhausen, I., Schachter, H. & Stanley, P. Essentials of Glycobiology 2nd edn. (Cold Spring Harbor Laboratory Press, 2009).

  55. 55

    Wisniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359 (2009).

    CAS  PubMed  Article  Google Scholar 

  56. 56

    Morelle, W. & Michalski, J.-C. Analysis of protein glycosylation by mass spectrometry. Nat. Protoc. 2, 1585–1602 (2007).

    CAS  PubMed  Article  Google Scholar 

  57. 57

    Wang, C., Fan, W., Zhang, P., Wang, Z. & Huang, L. One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-MS analysis. Proteomics 11, 4229–4242 (2011).

    PubMed  Article  CAS  Google Scholar 

  58. 58

    Sić, S., Maier, N.M. & Rizzi, A.M. Quantitative fingerprinting of O-linked glycans released from proteins using isotopic coded labeling with deuterated 1-phenyl-3-methyl-5-pyrazolone. J. Chromatogr. A 1408, 93–100 (2015).

    PubMed  Article  CAS  Google Scholar 

  59. 59

    Windwarder, M. & Altmann, F. Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry. J. Proteomics 108, 258–268 (2014).

    CAS  PubMed  Article  Google Scholar 

  60. 60

    Yang, S. et al. Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method. Clin. Proteomics 14, 1–12 (2017).

    Article  CAS  Google Scholar 

Download references

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).

Author information

Affiliations

Authors

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.

Corresponding authors

Correspondence to Shuang Yang or Hui Zhang.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing