Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS

  • Christopher Ashwood
  • Chi-Hung Lin
  • Morten Thaysen-Andersen
  • Nicolle H. Packer
Focus: Mass Spectrometry in Glycobiology and Related Fields: Research Article


Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers.

Graphical Abstract


Glycomics Diagnostic ions Skyline MS/MS 



This study was supported by an Australian Postgraduate Award scholarship. We thank M. P. C., Griffith University, for the useful discussions. We also thank the Skyline development team for creating and developing the Skyline software package. This research project was facilitated by access to the Australian Proteomics Analysis Facility (APAF) established under the Australian Government’s NCRIS program. C. A. is funded by an Australian Postgraduate Award. M.T.-A. was supported by a fellowship from the Cancer Institute NSW, Australia.

Supplementary material

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  1. 1.
    Lin, S., Kemmner, W., Grigull, S., Schlag, P.M.: Cell surface a2,6-sialylation affects adhesion of breast carcinoma cells. Exp. Cell Res. 110, 101–110 (2002)CrossRefGoogle Scholar
  2. 2.
    Zaia, J.: Mass spectrometry of oligosaccharides. Mass Spectrom. Rev. 23, 161–227 (2004)CrossRefGoogle Scholar
  3. 3.
    Abrahams, J.L., Campbell, M.P., Packer, N.H.: Building a PGC-LC-MS N-glycan retention library and elution mapping resource. Glycoconj. J. 1–15 Advance online publication. (2017)
  4. 4.
    Pabst, M., Bondili, J.S., Stadlmann, J., Mach, L., Altmann, F.: Mass + retention time = structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans. Anal. Chem. 79, 5051–5057 (2007)CrossRefGoogle Scholar
  5. 5.
    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, 622–630 (2005)CrossRefGoogle Scholar
  6. 6.
    Harvey, D.J.: Fragmentation of negative ions from carbohydrates: part 2. Fragmentation of high-mannose N-linked glycans. J. Am. Soc. Mass Spectrom. 16, 631–646 (2005)CrossRefGoogle Scholar
  7. 7.
    Harvey, D.J.: Fragmentation of negative ions from carbohydrates: part 3. Fragmentation of hybrid and complex N-linked glycans. J. Am. Soc. Mass Spectrom. 16, 647–659 (2005)CrossRefGoogle Scholar
  8. 8.
    Harvey, D.J., Jaeken, J., Butler, M., Armitage, A.J., Rudd, P., 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, 528–535 (2010)CrossRefGoogle Scholar
  9. 9.
    Harvey, D.J., Rudd, P.M.: Fragmentation of negative ions from N-linked carbohydrates. Part 5: anionic N-linked glycans. Int. J. Mass Spectrom. 305, 120–130 (2011)CrossRefGoogle Scholar
  10. 10.
    Harvey, D.J., Edgeworth, M., Krishna, B.A., Bonomelli, C., Allman, S.A., Crispin, M., Scrivens, J.H.: Fragmentation of negative ions from N-linked carbohydrates: part 6. Glycans containing one N-acetylglucosamine in the core. Rapid Commun. Mass Spectrom. 28, 2008–2018 (2014)CrossRefGoogle Scholar
  11. 11.
    Harvey, D.J., Abrahams, J.L.: Fragmentation and ion mobility properties of negative ions from N-linked carbohydrates: part 7. Reduced glycans. Rapid Commun. Mass Spectrom. 30, 627–634 (2016)CrossRefGoogle Scholar
  12. 12.
    Pfenninger, A., Karas, M., Finke, B., Stahl, B.: Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (part 1: methodology). J. Am. Soc. Mass Spectrom. 13, 1331–1340 (2002)CrossRefGoogle Scholar
  13. 13.
    Doohan, R.A., Hayes, C.A., Harhen, B., Karlsson, N.G.: Negative ion CID fragmentation of o-linked oligosaccharide aldoses-charge induced and charge remote fragmentation. J. Am. Soc. Mass Spectrom. 22, 1052–1062 (2011)CrossRefGoogle Scholar
  14. 14.
    Harvey, D.J., Martin, R.L., Jackson, K.A., Sutton, C.W.: Fragmentation of N-linked glycans with a matrix-assisted laser desorption/ionization ion trap time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 18, 2997–3007 (2004)CrossRefGoogle Scholar
  15. 15.
    Bereman, M.S., Canterbury, J.D., Egertson, J.D., Horner, J., Remes, P.M., Schwartz, J.C., Zabrouskov, V., Maccoss, M.J.: Evaluation of front-end higher energy collision-induced dissociation on a bench-top dual pressure linear ion trap mass spectrometer for shotgun proteomics. Anal. Chem. 84, 1533–1539 (2012)CrossRefGoogle Scholar
  16. 16.
    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
  17. 17.
    Everest-Dass, A.V., Abrahams, J.L., Kolarich, D., Packer, N.H., Campbell, M.P.: Structural feature ions for distinguishing N- and O-linked glycan isomers by LC-ESI-IT MS/MS. J. Am. Soc. Mass Spectrom. 24, 895–906 (2013)CrossRefGoogle Scholar
  18. 18.
    Wheeler, S.F., Harvey, D.J.: Negative ion mass spectrometry of sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by MALDI-TOF and ESI-TOF mass spectrometry. Anal. Chem. 72, 5027–5039 (2000)CrossRefGoogle Scholar
  19. 19.
    Spina, L., Romeo, D., Impallomeni, G., Garozzo, D., Waidelich, D., Glueckmann, M.: New fragmentation mechanisms in matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates. Rapid Commun. Mass Spectrom. 18, 392–398 (2004)CrossRefGoogle Scholar
  20. 20.
    Ranzinger, R., Weatherly, D., Arpinar, S., Khan, S., Porterfield, M., Tiemeyer, M., S. W. York: GRITS Toolbox—a software system for the archival, processing and interpretation of glycomics data. In: Annual Meeting of the Society-for-Glycobiology on Glycobiology. p. 1275 (2015)Google Scholar
  21. 21.
    Yu, C.Y., Mayampurath, A., Hu, Y., Zhou, S., Mechref, Y., Tang, H.: Automated annotation and quantification of glycans using liquid chromatography-mass spectrometry. Bioinformatics. 29, 1706–1707 (2013)CrossRefGoogle Scholar
  22. 22.
    MacLean, B., Tomazela, D.M., Shulman, N., Chambers, M., Finney, G.L., Frewen, B., Kern, R., Tabb, D.L., Liebler, D.C., MacCoss, M.J.: Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 26, 966–968 (2010)CrossRefGoogle Scholar
  23. 23.
    Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976)CrossRefGoogle Scholar
  24. 24.
    Jensen, P.H., Karlsson, N.G., Kolarich, D., Packer, N.H.: Structural analysis of N- and O-glycans released from glycoproteins. Nat. Protoc. 7, 1299–1310 (2012)CrossRefGoogle Scholar
  25. 25.
    Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P.: ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics. 24, 2534–2536 (2008)CrossRefGoogle Scholar
  26. 26.
    Nagy, G., Pohl, N.L.B.: Monosaccharide identification as a first step toward de novo carbohydrate sequencing: mass spectrometry strategy for the identification and differentiation of diastereomeric and enantiomeric pentose isomers. Anal. Chem. 87, 4566–4571 (2015)CrossRefGoogle Scholar
  27. 27.
    Webb, I.K., Chen, T.C., Danielson, W.F., Ibrahim, Y.M., Tang, K., Anderson, G.A., Smith, R.D.: Implementation of dipolar resonant excitation for collision induced dissociation with ion mobility/time-of-flight MS. J. Am. Soc. Mass Spectrom. 25, 563–571 (2014)CrossRefGoogle Scholar
  28. 28.
    Everest-Dass, A.V., Jin, D., Thaysen-Andersen, M., Nevalainen, H., Kolarich, D., Packer, N.H.: Comparative structural analysis of the glycosylation of salivary and buccal cell proteins: innate protection against infection by Candida albicans. Glycobiology. 22, 1465–1479 (2012)CrossRefGoogle Scholar
  29. 29.
    Harvey, D.J., Royle, L., Radcliffe, C.M., Rudd, P.M., Dwek, R.A.: Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry. Anal. Biochem. 376, 44–60 (2008)CrossRefGoogle Scholar
  30. 30.
    Mcclellan, J.E., Costello, C.E., Connor, P.B.O., Zaia, J.: Influence of charge state on product ion mass spectra and the determination of 4S / 6S sulfation sequence of chondroitin sulfate oligosaccharides. Anal. Chem. 74, 3760–3771 (2002)CrossRefGoogle Scholar
  31. 31.
    Brockhausen, I.: Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Rep. 7, 599–604 (2006)CrossRefGoogle Scholar
  32. 32.
    Yu, X., Huang, Y., Lin, C., Costello, C.E.: Energy-dependent electron activated dissociation of metal-adducted permethylated oligosaccharides. Anal. Chem. 84, 7487–7494 (2012)CrossRefGoogle Scholar
  33. 33.
    Bythell, B.J., Abutokaikah, M.T., Wagoner, A.R., Guan, S., Rabus, J.M.: Cationized carbohydrate gas-phase fragmentation chemistry. J. Am. Soc. Mass Spectrom. 28, 688–703 (2017)CrossRefGoogle Scholar
  34. 34.
    Chen, C.-H., Lin, Y.-P., Lin, J.-L., Li, S.-T., Ren, C.-T., Wu, C.-Y., Chen, C.-H.: Rapid identification of terminal sialic acid linkage isomers by pseudo-MS3 mass spectrometry. Isr. J. Chem. 55, 412–422 (2015)CrossRefGoogle Scholar
  35. 35.
    Loziuk, P.L., Hecht, E.S., Muddiman, D.C.: N-linked glycosite profiling and use of Skyline as a platform for characterization and relative quantification of glycans in differentiating xylem of Populus trichocarpa. Anal. Bioanal. Chem. 409, 487–497 (2017)CrossRefGoogle Scholar
  36. 36.
    Pabst, M., Altmann, F.: Influence of electrosorption, solvent, temperature, and ion polarity on the performance of LC-ESI-MS using graphitic carbon for acidic oligosaccharides. Anal. Chem. 80, 7534–7542 (2008)CrossRefGoogle Scholar
  37. 37.
    Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., Dwek, R.A.: A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240, 210–226 (1996)CrossRefGoogle Scholar
  38. 38.
    Sharma, V., Eckels, J., Taylor, G.K., Shulman, N.J., Stergachis, A.B., Joyner, S.A., Yan, P., Whiteaker, J.R., Halusa, G.N., Schilling, B., Gibson, B.W., Colangelo, C.M., Paulovich, A.G., Carr, S.A., Jaffe, J.D., Maccoss, M.J., Maclean, B.: Panorama: a targeted proteomics knowledge base. J. Proteome Res. 13, 4205–4210 (2014)CrossRefGoogle Scholar
  39. 39.
    Loo, J.A., Matthews, D.E., Yates, J.R.: Focus on bioinformatics, software, and MS-based “Omics,” honoring Dr. Michael J. MacCoss, recipient of the 2015 ASMS Biemann medal. J. Am. Soc. Mass Spectrom. 27, 1715–1718 (2016)CrossRefGoogle Scholar
  40. 40.
    Kolarich, D., Rapp, E., Struwe, W.B., Haslam, S.M., Zaia, J., McBride, R., Agravat, S., Campbell, M.P., Kato, M., Ranzinger, R., Kettner, C., York, W.S.: The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting mass-spectrometry-based glycoanalytic data. Mol. Cell. Proteomics. 12, 991–995 (2013)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of Molecular SciencesMacquarie UniversitySydneyAustralia
  2. 2.Australian Research Council Centre of Excellence for Nanoscale BiophotonicsMacquarie UniversitySydneyAustralia
  3. 3.Australian Proteome Analysis FacilityMacquarie UniversitySydneyAustralia
  4. 4.Institute for GlycomicsGriffith UniversitySouthportAustralia

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