Negative Electron Transfer Dissociation Sequencing of 3-O-Sulfation-Containing Heparan Sulfate Oligosaccharides

  • Jiandong Wu
  • Juan Wei
  • John D. Hogan
  • Pradeep Chopra
  • Apoorva Joshi
  • Weigang Lu
  • Joshua Klein
  • Geert-Jan Boons
  • Cheng Lin
  • Joseph Zaia
Focus: Mass Spectrometry in Glycobiology and Related Fields: Research Article


Among dissociation methods, negative electron transfer dissociation (NETD) has been proven the most useful for glycosaminoglycan (GAG) sequencing because it produces informative fragmentation, a low degree of sulfate losses, high sensitivity, and translatability to multiple instrument types. The challenge, however, is to distinguish positional sulfation. In particular, NETD has been reported to fail to differentiate 4-O- versus 6-O-sulfation in chondroitin sulfate decasaccharide. This raised the concern of whether NETD is able to differentiate the rare 3-O-sulfation from predominant 6-O-sulfation in heparan sulfate (HS) oligosaccharides. Here, we report that NETD generates highly informative spectra that differentiate sites of O-sulfation on glucosamine residues, enabling structural characterizations of synthetic HS isomers containing 3-O-sulfation. Further, lyase-resistant 3-O-sulfated tetrasaccharides from natural sources were successfully sequenced. Notably, for all of the oligosaccharides in this study, the successful sequencing is based on NETD tandem mass spectra of commonly observed deprotonated precursor ions without derivatization or metal cation adduction, simplifying the experimental workflow and data interpretation. These results demonstrate the potential of NETD as a sensitive analytical tool for detailed, high-throughput structural analysis of highly sulfated GAGs.

Graphical Abstract


Negative electron transfer dissociation Fourier transform ion cyclotron resonance mass spectrometry Glycosaminoglycan Heparan sulfate Sulfation Glycomics 


Funding Information

This work was supported by NIH grants P41GM104603, R21HL131554, and U01CA221234.

Supplementary material

13361_2018_1907_MOESM1_ESM.pdf (1.8 mb)
ESM 1 (PDF 1849 kb)


  1. 1.
    Knelson, E.H., Nee, J.C., Blobe, G.C.: Heparan sulfate signaling in cancer. Trends Biochem. Sci. 39, 277–288 (2014)CrossRefGoogle Scholar
  2. 2.
    Esko, J.D., Selleck, S.B.: Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435–471 (2002)CrossRefGoogle Scholar
  3. 3.
    Thacker, B.E., Seamen, E., Lawrence, R., Parker, M.W., Xu, Y., Liu, J., Vander Kooi, C.W., Esko, J.D.: Expanding the 3-O-sulfate proteome-enhanced binding of neuropilin-1 to 3-O-sulfated heparan sulfate modulates its activity. ACS Chem. Biol. 11, 971–980 (2016)CrossRefGoogle Scholar
  4. 4.
    Patel, V.N., Lombaert, I.M., Cowherd, S.N., Shworak, N.W., Xu, Y., Liu, J., Hoffman, M.P.: Hs3st3-modified heparan sulfate controls KIT+ progenitor expansion by regulating 3-O-sulfotransferases. Dev. Cell. 29, 662–673 (2014)CrossRefGoogle Scholar
  5. 5.
    Esko, J.D., Lindahl, U.: Molecular diversity of heparan sulfate. J. Clin. Invest. 108, 169–173 (2001)CrossRefGoogle Scholar
  6. 6.
    Marcum, J.A., Atha, D.H., Fritze, L.M., Nawroth, P., Stern, D., Rosenberg, R.D.: Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J. Biol. Chem. 261, 7507–7517 (1986)Google Scholar
  7. 7.
    Pejler, G., Danielsson, A., Bjork, I., Lindahl, U., Nader, H.B., Dietrich, C.P.: Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides. J. Biol. Chem. 262, 11413–11421 (1987)Google Scholar
  8. 8.
    de Agostini, A.I., Dong, J.C., de Vantery Arrighi, C., Ramus, M.A., Dentand-Quadri, I., Thalmann, S., Ventura, P., Ibecheole, V., Monge, F., Fischer, A.M., HajMohammadi, S., Shworak, N.W., Zhang, L., Zhang, Z., Linhardt, R.J.: Human follicular fluid heparan sulfate contains abundant 3-O-sulfated chains with anticoagulant activity. J. Biol. Chem. 283, 28115–28124 (2008)CrossRefGoogle Scholar
  9. 9.
    Li, G., Yang, B., Li, L., Zhang, F., Xue, C., Linhardt, R.J.: Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography-mass spectrometry. Anal. Biochem. 455, 3–9 (2014)CrossRefGoogle Scholar
  10. 10.
    Li, G., Steppich, J., Wang, Z., Sun, Y., Xue, C., Linhardt, R.J., Li, L.: Bottom-up low molecular weight heparin analysis using liquid chromatography-Fourier transform mass spectrometry for extensive characterization. Anal. Chem. 86, 6626–6632 (2014)CrossRefGoogle Scholar
  11. 11.
    Liu, J., Pedersen, L.C.: Anticoagulant heparan sulfate: structural specificity and biosynthesis. Appl. Microbiol. Biotechnol. 74, 263–272 (2007)CrossRefGoogle Scholar
  12. 12.
    Thacker, B.E., Xu, D., Lawrence, R., Esko, J.D.: Heparan sulfate 3-O-sulfation: a rare modification in search of a function. Matrix Biol. 35, 60–72 (2014)CrossRefGoogle Scholar
  13. 13.
    Liu, J., Shworak, N.W., Fritze, L.M., Edelberg, J.M., Rosenberg, R.D.: Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 271, 27072–27082 (1996)CrossRefGoogle Scholar
  14. 14.
    Shukla, D., Liu, J., Blaiklock, P., Shworak, N.W., Bai, X., Esko, J.D., Cohen, G.H., Eisenberg, R.J., Rosenberg, R.D., Spear, P.G.: A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell. 99, 13–22 (1999)CrossRefGoogle Scholar
  15. 15.
    Tiwari, V., O’Donnell, C.D., Oh, M.J., Valyi-Nagy, T., Shukla, D.: A role for 3-O-sulfotransferase isoform-4 in assisting HSV-1 entry and spread. Biochem. Biophys. Res. Commun. 338, 930–937 (2005)CrossRefGoogle Scholar
  16. 16.
    Xu, D., Tiwari, V., Xia, G., Clement, C., Shukla, D., Liu, J.: Characterization of heparan sulphate 3-O-sulphotransferase isoform 6 and its role in assisting the entry of herpes simplex virus type 1. Biochem. J. 385, 451–459 (2005)CrossRefGoogle Scholar
  17. 17.
    O’Donnell, C.D., Tiwari, V., Oh, M.J., Shukla, D.: A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread. Virology. 346, 452–459 (2006)CrossRefGoogle Scholar
  18. 18.
    Zaia, J.: Glycosaminoglycan glycomics using mass spectrometry. Mol. Cell. Proteomics. 12, 885–892 (2013)CrossRefGoogle Scholar
  19. 19.
    Zaia, J.: Mass spectrometry of oligosaccharides. Mass Spectrom. Rev. 23, 161–227 (2004)CrossRefGoogle Scholar
  20. 20.
    Wolff, J.J., Chi, L., Linhardt, R.J., Amster, I.J.: Distinguishing glucuronic from iduronic acid in glycosaminoglycan tetrasaccharides by using electron detachment dissociation. Anal. Chem. 79, 2015–2022 (2007)CrossRefGoogle Scholar
  21. 21.
    Wolff, J.J., Laremore, T.N., Busch, A.M., Linhardt, R.J., Amster, I.J.: Influence of charge state and sodium cationization on the electron detachment dissociation and infrared multiphoton dissociation of glycosaminoglycan oligosaccharides. J. Am. Soc. Mass Spectrom. 19, 790–798 (2008)CrossRefGoogle Scholar
  22. 22.
    Wolff, J.J., Laremore, T.N., Busch, A.M., Linhardt, R.J., Amster, I.J.: Electron detachment dissociation of dermatan sulfate oligosaccharides. J. Am. Soc. Mass Spectrom. 19, 294–304 (2008)CrossRefGoogle Scholar
  23. 23.
    Wolff, J.J., Leach 3rd, F.E., Laremore, T.N., Kaplan, D.A., Easterling, M.L., Linhardt, R.J., Amster, I.J.: Negative electron transfer dissociation of glycosaminoglycans. Anal. Chem. 82, 3460–3466 (2010)CrossRefGoogle Scholar
  24. 24.
    Leach, F. E. 3rd; Arungundram, S.; Al-Mafraji, K.; Venot, A.; Boons, G. J.; Amster, I. J. Electron detachment dissociation of synthetic heparan sulfate glycosaminoglycan tetrasaccharides varying in degree of sulfation and hexuronic acid stereochemistry. Int. J. Mass Spectrom., 330–332, 152–159 (2012)Google Scholar
  25. 25.
    Leach 3rd, F.E., Ly, M., Laremore, T.N., Wolff, J.J., Perlow, J., Linhardt, R.J., Amster, I.J.: Hexuronic acid stereochemistry determination in chondroitin sulfate glycosaminoglycan oligosaccharides by electron detachment dissociation. J. Am. Soc. Mass Spectrom. 23, 1488–1497 (2012)CrossRefGoogle Scholar
  26. 26.
    Leach 3rd, F.E., Wolff, J.J., Xiao, Z., Ly, M., Laremore, T.N., Arungundram, S., Al-Mafraji, K., Venot, A., Boons, G.J., Linhardt, R.J., Amster, I.J.: Negative electron transfer dissociation Fourier transform mass spectrometry of glycosaminoglycan carbohydrates. Eur J Mass Spectrom (Chichester). 17, 167–176 (2011)CrossRefGoogle Scholar
  27. 27.
    Leach 3rd, F.E., Xiao, Z., Laremore, T.N., Linhardt, R.J., Amster, I.J.: Electron detachment dissociation and infrared multiphoton dissociation of heparin tetrasaccharides. Int. J. Mass Spectrom. 308, 253–259 (2011)CrossRefGoogle Scholar
  28. 28.
    Huang, Y., Yu, X., Mao, Y., Costello, C.E., Zaia, J., Lin, C.: De novo sequencing of heparan sulfate oligosaccharides by electron-activated dissociation. Anal. Chem. 85, 11979–11986 (2013)CrossRefGoogle Scholar
  29. 29.
    Agyekum, I., Zong, C., Boons, G.J., Amster, I.J.: Single stage tandem mass spectrometry assignment of the C-5 uronic acid stereochemistry in heparan sulfate tetrasaccharides using electron detachment dissociation. J. Am. Soc. Mass Spectrom. (2017)Google Scholar
  30. 30.
    Agyekum, I., Patel, A.B., Zong, C., Boons, G.J., Amster, J.: Assignment of hexuronic acid stereochemistry in synthetic heparan sulfate tetrasaccharides with 2-O-sulfo uronic acids using electron detachment dissociation. Int. J. Mass Spectrom. 390, 163–169 (2015)CrossRefGoogle Scholar
  31. 31.
    Oh, H.B., Leach 3rd, F.E., Arungundram, S., Al-Mafraji, K., Venot, A., Boons, G.J., Amster, I.J.: Multivariate analysis of electron detachment dissociation and infrared multiphoton dissociation mass spectra of heparan sulfate tetrasaccharides differing only in hexuronic acid stereochemistry. J. Am. Soc. Mass Spectrom. 22, 582–590 (2011)CrossRefGoogle Scholar
  32. 32.
    Zaia, J., Li, X.Q., Chan, S.Y., Costello, C.E.: Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides. J. Am. Soc. Mass Spectrom. 14, 1270–1281 (2003)CrossRefGoogle Scholar
  33. 33.
    Miller, M.J., Costello, C.E., Malmstrom, A., Zaia, J.: A tandem mass spectrometric approach to determination of chondroitin/dermatan sulfate oligosaccharide glycoforms. Glycobiology. 16, 502–513 (2006)CrossRefGoogle Scholar
  34. 34.
    Leach 3rd, F.E., Riley, N.M., Westphall, M.S., Coon, J.J., Amster, I.J.: Negative electron transfer dissociation sequencing of increasingly sulfated glycosaminoglycan oligosaccharides on an Orbitrap mass spectrometer. J. Am. Soc. Mass Spectrom. (2017)Google Scholar
  35. 35.
    Chiu, Y., Huang, R., Orlando, R., Sharp, J.S.: GAG-ID: heparan sulfate (HS) and heparin glycosaminoglycan high-throughput identification software. Mol. Cell. Proteomics. 14, 1720–1730 (2015)CrossRefGoogle Scholar
  36. 36.
    Huang, R., Zong, C., Venot, A., Chiu, Y., Zhou, D., Boons, G.J., De Sharp, J.S.: Novo sequencing of complex mixtures of heparan sulfate oligosaccharides. Anal. Chem. 88, 5299–5307 (2016)CrossRefGoogle Scholar
  37. 37.
    Huang, R., Liu, J., Sharp, J.S.: An approach for separation and complete structural sequencing of heparin/heparan sulfate-like oligosaccharides. Anal. Chem. 85, 5787–5795 (2013)CrossRefGoogle Scholar
  38. 38.
    Arungundram, S., Al-Mafraji, K., Asong, J., Leach 3rd, F.E., Amster, I.J., Venot, A., Turnbull, J.E., Boons, G.J.: Modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies. J. Am. Chem. Soc. 131, 17394–17405 (2009)CrossRefGoogle Scholar
  39. 39.
    Zaia, J., Khatri, K., Klein, J., Shao, C., Sheng, Y., Viner, R.: Complete molecular weight profiling of low-molecular weight heparins using size exclusion chromatography-ion suppressor-high-resolution mass spectrometry. Anal. Chem. 88, 10654–10660 (2016)CrossRefGoogle Scholar
  40. 40.
    Huang, Y., Mao, Y., Zong, C., Lin, C., Boons, G.J., Zaia, J.: Discovery of a heparan sulfate 3-O-sulfation specific peeling reaction. Anal. Chem. 87, 592–600 (2015)CrossRefGoogle Scholar
  41. 41.
    Damerell, D., Ceroni, A., Maass, K., Ranzinger, R., Dell, A., Haslam, S.M.: The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments. Biol. Chem. 393, 1357–1362 (2012)CrossRefGoogle Scholar
  42. 42.
    Domon, B., Costello, C.E.: A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409 (1988)CrossRefGoogle Scholar
  43. 43.
    Huang, Y., Shi, X., Yu, X., Leymarie, N., Staples, G.O., Yin, H., Killeen, K., Zaia, J.: Improved liquid chromatography-MS/MS of heparan sulfate oligosaccharides via chip-based pulsed makeup flow. Anal. Chem. 83, 8222–8229 (2011)CrossRefGoogle Scholar
  44. 44.
    Shi, X., Huang, Y., Mao, Y., Naimy, H., Zaia, J.: Tandem mass spectrometry of heparan sulfate negative ions: sulfate loss patterns and chemical modification methods for improvement of product ion profiles. J. Am. Soc. Mass Spectrom. 23, 1498–1511 (2012)CrossRefGoogle Scholar
  45. 45.
    Zaia, J., Costello, C.E.: Tandem mass spectrometry of sulfated heparin-like glycosaminoglycan oligosaccharides. Anal. Chem. 75, 2445–2455 (2003)CrossRefGoogle Scholar
  46. 46.
    Chen, Y., Lin, L., Agyekum, I., Zhang, X., St Ange, K., Yu, Y., Zhang, F., Liu, J., Amster, I.J., Linhardt, R.J.: Structural analysis of heparin-derived 3-O-sulfated tetrasaccharides: antithrombin binding site variants. J. Pharm. Sci. 106, 973–981 (2017)CrossRefGoogle Scholar
  47. 47.
    Fu, L., Li, G., Yang, B., Onishi, A., Li, L., Sun, P., Zhang, F., Linhardt, R.J.: Structural characterization of pharmaceutical heparins prepared from different animal tissues. J. Pharm. Sci. 102, 1447–1457 (2013)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Center for Biomedical Mass Spectrometry, Department of Biochemistry and Center for Biomedical Mass SpectrometryBoston University School of MedicineBostonUSA
  2. 2.Bioinformatics ProgramBoston UniversityBostonUSA
  3. 3.Complex Carbohydrate Research CenterUniversity of GeorgiaAthensUSA
  4. 4.Department of ChemistryUniversity of GeorgiaAthensUSA
  5. 5.Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Bijvoet Center for Biomolecular ResearchUtrecht UniversityUtrechtNetherlands

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