Simple Approach for De Novo Structural Identification of Mannose Trisaccharides

  • Hsu Chen Hsu
  • Chia Yen Liew
  • Shih-Pei Huang
  • Shang-Ting Tsai
  • Chi-Kung Ni
Research Article


Oligosaccharides have diverse functions in biological systems. However, the structural determination of oligosaccharides remains difficult and has created a bottleneck in carbohydrate research. In this study, a new approach for the de novo structural determination of underivatized oligosaccharides is demonstrated. A low-energy collision-induced dissociation (CID) of sodium ion adducts was used to facilitate the cleavage of desired chemical bonds during the dissociation. The selection of fragments for the subsequent CID was guided using a procedure that we built from the understanding of the saccharide dissociation mechanism. The linkages, anomeric configurations, and branch locations of oligosaccharides were determined by comparing the CID spectra of oligosaccharide with the fragmentation patterns based on the dissociation mechanism and our specially prepared disaccharide CID spectrum database. The usefulness of this method was demonstrated to determine the structures of several mannose trisaccharides. This method can also be applied in the structural determination of oligosaccharides larger than trisaccharides and containing hexose other than mannose if authentic standards are available.

Graphical Abstract


Oligosaccharide Structure CID 



This work was financially supported in part by the Ministry of Science and Technology, Taiwan (103-2113-M-001-011-NY3).

Supplementary material

13361_2017_1850_MOESM1_ESM.docx (84 kb)
ESM 1 (DOCX 84 kb)


  1. 1.
    Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W., Etzler, M.E. (eds.): Essentials of Glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2009)Google Scholar
  2. 2.
    Bertozzi, C.R., Kiessling, L.L.: Chemical glycobiology. Science. 291(5512), 2357–2364 (2001)CrossRefGoogle Scholar
  3. 3.
    National Research Council: Transforming Glycoscience: A Roadmap for the Future. The National Academies Press, Washington, DC (2012)Google Scholar
  4. 4.
    Laine, R.A.: A Calculation of All Possible Oligosaccharide Isomers Both Branched and Linear Yields 1.05x10(12) Structures for a Reducing Hexasaccharide - the Isomer-Barrier to Development of Single-Method Saccharide Sequencing or Synthesis Systems. Glycobiology. 4(6), 759–767 (1994)CrossRefGoogle Scholar
  5. 5.
    Duus, J.O., Gotfredsen, C.H., Bock, K.: Chem. Rev. 100, 4589 (2000)CrossRefGoogle Scholar
  6. 6.
    Kailemia, M.J., Ruhaak, L.R., Lebrilla, C.B., Amster, I.J.: Oligosaccharide analysis by mass spectrometry: a review of recent developments. Anal. Chem. 86(1), 196–212 (2014)CrossRefGoogle Scholar
  7. 7.
    Naven, T.J., Harvey, D.J.: Effect of structure on the signal strength of oligosaccharides in matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments. Rapid Commun. Mass Spectrom. 10(11), 1361–1366 (1996)CrossRefGoogle Scholar
  8. 8.
    Chen, J.L., Lee, C., Lu, I.C., Chien, C.L., Lee, Y.T., Hu, W.P., Ni, C.K.: Theoretical investigation of low detection sensitivity for underivatized carbohydrates in ESI and MALDI. J. Mass Spectrom. 51(12), 1180–1186 (2016)Google Scholar
  9. 9.
    Zaia, J.: Mass spectrometry of oligosaccharides. Mass Spectrom. Rev. 23(3), 161–227 (2004)CrossRefGoogle Scholar
  10. 10.
    Stephens, E., Maslen, S.L., Green, L.G., Williams, D.H.: Fragmentation characteristics of neutral N-linked glycans using a MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 76(8), 2343–2354 (2004)CrossRefGoogle Scholar
  11. 11.
    Kurimoto, A., Daikoku, S., Mutsuga, S., Kanie, O.: Analysis of energy-resolved mass spectra at MS n in a pursuit to characterize structural isomers of oligosaccharides. Anal. Chem. 78(10), 3461–3466 (2006)CrossRefGoogle Scholar
  12. 12.
    Vijayakrishnan, B., Issaree, A., Corilo, Y.E., Ferreira, C.R., Eberlin, M.N., Peter, M.G.: MSn of the six isomers of (GlcN) 2(GlcNAc) 2aminoglucan tetrasaccharides (diacetylchitotetraoses): Rules of fragmentation for the sodiated molecules and application to sequence analysis of hetero-chitooligosaccharides. Carbohydr. Polym. 84(2), 713–726 (2011)Google Scholar
  13. 13.
    Li, D.T., Her, G.R.: Structural analysis of chromophore-labeled disaccharides and oligosaccharides by electrospray ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry. J. Mass Spectrom. 33(7), 644–652 (1998)CrossRefGoogle Scholar
  14. 14.
    Cheng, H.L., Her, G.R.: Determination of linkages of linear and branched oligosaccharides using closed-ring chromophore labeling and negative ion trap mass spectrometry. J. Am. Soc. Mass. Spectrom. 13(11), 1322–1330 (2002)CrossRefGoogle Scholar
  15. 15.
    Harvey, D.J.: Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates. J. Am. Soc. Mass. Spectrom. 16(5), 622–630 (2005)CrossRefGoogle Scholar
  16. 16.
    Harvey, D.J.: Fragmentation of negative ions from carbohydrates: part 2. Fragmentation of high-mannose N-linked glycans. J. Am. Soc. Mass Spectrom. 16(5), 631–646 (2005)CrossRefGoogle Scholar
  17. 17.
    Guan, B., Cole, R.B.: MALDI linear-field reflectron TOF post-source decay analysis of underivatized oligosaccharides: Determination of glycosidic linkages and anomeric configurations using anion attachment. J. Am. Soc. Mass. Spectrom. 19(8), 1119–1131 (2008)CrossRefGoogle Scholar
  18. 18.
    Harvey, D.J., Jaeken, J., Butler, M., Armitage, A.J., Rudd, P.M., Dwek, R.A.: Fragmentation of negative ions from N-linked carbohydrates, part 4. Fragmentation of complex glycans lacking substitution on the 6-antenna. J. Mass Spectrom. 45(5), 528–535 (2010)CrossRefGoogle Scholar
  19. 19.
    Viseux, N., de Hoffmann, E., Domon, B.: Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry. Anal. Chem. 70(23), 4951–4959 (1998)CrossRefGoogle Scholar
  20. 20.
    van der Kerk, S.M., Blok-Tip, L., van der Kerk-van Hoof, A., Heerma, W., Haverkamp, J.: Differences in fragmentation behaviour between α-and β-linked derivatized xylobiosides: explanation in terms of sigma conjugation. Int. J. Mass Spectrom. Ion Processes. 134(1), 41–54 (1994)CrossRefGoogle Scholar
  21. 21.
    Xue, J., Song, L., Khaja, S.D., Locke, R.D., West, C.M., Laine, R.A., Matta, K. L.: Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 18(17), 1947–1955 (2004)Google Scholar
  22. 22.
    Mendonca, S., Cole, R.B., Zhu, J., Cai, Y., French, A.D., Johnson, G.P., Laine R.A.: Incremented alkyl derivatives enhance collision induced glycosidic bond cleavage in mass spectrometry of disaccharides. J. Am. Soc. Mass. Spectrom. 14(1), 63–78 (2003)Google Scholar
  23. 23.
    Ashline, D., Singh, S., Hanneman, A., Reinhold, V.: Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MS n. Anal. Chem. 77(19), 6250–6262 (2005)CrossRefGoogle Scholar
  24. 24.
    Zhang, H., Singh, S., Reinhold, V.N.: Congruent strategies for carbohydrate sequencing. 2. FragLib: An MS n spectral library. Anal. Chem. 77(19), 6263–6270 (2005)CrossRefGoogle Scholar
  25. 25.
    Nagy, G., Pohl, N.L.: Complete hexose isomer identification with mass spectrometry. J. Am. Soc. Mass. Spectrom. 26(4), 677–685 (2015)CrossRefGoogle Scholar
  26. 26.
    Fang, T.T., Zirrolli, J., Bendiak, B.: Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards. Carbohydr. Res. 342(2), 217–235 (2007)Google Scholar
  27. 27.
    Fang, T.T., Bendiak, B.: The stereochemical dependence of unimolecular dissociation of monosaccharide-glycolaldehyde anions in the gas phase: a basis for assignment of the stereochemistry and anomeric configuration of monosaccharides in oligosaccharides by mass spectrometry via a key discriminatory product ion of disaccharide fragmentation, m/z 221. J. Am. Chem. Soc. 129(31), 9721–9736 (2007)Google Scholar
  28. 28.
    Konda, C., Londry, F.A., Bendiak, B., Xia, Y.: Assignment of the Stereochemistry and Anomeric Configuration of Sugars within Oligosaccharides Via Overlapping Disaccharide Ladders Using MSn. J. Am. Soc. Mass. Spectrom. 25(8), 1441–1450 (2014)CrossRefGoogle Scholar
  29. 29.
    Ni, C.K.: Simple methods for de novo structural determination of glucose-containing underivatized oligosaccharides. In the division of carbohydrate chemistry, 254th American Chemical Society National Meeting & Exposition August 20-24, 2017, Washington, DC USAGoogle Scholar
  30. 30.
    Konda, C., Bendiak, B., Xia, Y.: Differentiation of the stereochemistry and anomeric configuration for 1-3 linked disaccharides via tandem mass spectrometry and 18O-labeling. J. Am. Soc. Mass. Spectrom. 23(2), 347–358 (2012)CrossRefGoogle Scholar
  31. 31.
    Hofmeister, G.E., Zhou, Z., Leary, J.A.: Linkage Position Determination in Lithium-Cationized Disaccharides - Tandem Mass-Spectrometry and Semiempirical Calculations. J. Am. Chem. Soc. 113(16), 5964–5970 (1991)CrossRefGoogle Scholar
  32. 32.
    Asam, M.R., Glish, G.L.: Tandem mass spectrometry of alkali cationized polysaccharides in a quadrupole ion trap. J. Am. Soc. Mass. Spectrom. 8(9), 987–995 (1997)CrossRefGoogle Scholar
  33. 33.
    da Costa, E.V., Moreira, A.S., Nunes, F.M., Coimbra, M.A., Evtuguin, D.V., Domingues, M.R.: Differentiation of isomeric pentose disaccharides by electrospray ionization tandem mass spectrometry and discriminant analysis. Rapid Commun. Mass Spectrom. 26(24), 2897–2904 (2012)CrossRefGoogle Scholar
  34. 34.
    Zhang, H., Brokman, S.M., Fang, N., Pohl, N.L., Yeung, E.S.: Linkage position and residue identification of disaccharides by tandem mass spectrometry and linear discriminant analysis. Rapid Commun. Mass Spectrom. 22(10), 1579–1586 (2008)CrossRefGoogle Scholar
  35. 35.
    Chen, J.L., Nguan, H.S., Hsu, P.J., Tsai, S.T., Liew, C.Y., Kuo, J.L., Hu, W.P., Ni, C.K.: Collision-induced dissociation of sodiated glucose and identification of anomeric configuration. Phys. Chem. Chem. Phys. 19, 15454–15462 (2017)Google Scholar

Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  1. 1.Institute of Atomic and Molecular SciencesAcademia SinicaTaipeiTaiwan
  2. 2.Department of ChemistryNational Taiwan Normal UniversityTaipeiTaiwan
  3. 3.Department of ChemistryNational Tsing Hua UniversityHsinchuTaiwan

Personalised recommendations