High-Field Asymmetric-Waveform Ion Mobility Spectrometry and Electron Detachment Dissociation of Isobaric Mixtures of Glycosaminoglycans

  • Muchena J. Kailemia
  • Melvin Park
  • Desmond A. Kaplan
  • Andre Venot
  • Geert-Jan Boons
  • Lingyun Li
  • Robert J. Linhardt
  • I. Jonathan Amster
Research Article


High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4–10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.

Key words

FAIMS Electron detachment dissociation Glycosaminoglycans Fourier transform ion cyclotron resonance mass spectrometry Differential mobility spectrometry Fourier transform mass spectrometry Carbohydrates 



M.J.K., L.L., R.J.L., and I.J.A. gratefully acknowledge financial support from the National Institutes of Health, R01-GM038060. M.J.K., A.V., G.J.B., and I.J.A. gratefully acknowledge financial support from the National Institutes of Health, P41-GM103390. M.J.K. personally acknowledges Dr. Franklin Leach for useful discussions about mass spectrometry analysis of GAGs.

Supplementary material

13361_2013_771_MOESM1_ESM.doc (246 kb)
ESM 1 (DOC 246 kb)


  1. 1.
    Vynios, D.H., Karamanos, N.K., Tsiganos, C.P.: Advances in analysis of glycosaminoglycans: its application for the assessment of physiological and pathological states of connective tissues. J. Chromatogr. B 781, 21–38 (2002)CrossRefGoogle Scholar
  2. 2.
    Capila, I., Linhardt, R.J.: Heparin–protein interactions. Angew. Chem. Int. Ed. 41, 390–412 (2002)CrossRefGoogle Scholar
  3. 3.
    Hardingham, T., Fosang, A.: Proteoglycans: many forms and many functions. The FASEB J. 6, 861–870 (1992)Google Scholar
  4. 4.
    Gandhi, N.S., Mancera, R.L.: The structure of glycosaminoglycans and their interactions with proteins. Chem. Biol. Drug Des 72, 455–482 (2008)CrossRefGoogle Scholar
  5. 5.
    Bernfield, M., Götte, M.; Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., Zako, M.: Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777 (1999)CrossRefGoogle Scholar
  6. 6.
    Lortat-Jacob, H., Grosdidier, A., Imberty, A.: Structural diversity of heparan sulfate binding domains in chemokines. Proc. Natl. Acad. Sci. 99, 1229–1234 (2002)CrossRefGoogle Scholar
  7. 7.
    Gupta, P., McCarthy, J., Verfaillie, C.: Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells. Blood 87, 3229–3236 (1996)Google Scholar
  8. 8.
    Ly, M., Leach F.E. III, Laremore, T.N., Toida, T., Amster, I.J., Linhardt, R. J.: The proteoglycan bikunin has a defined sequence. Nat. Chem. Biol. 7, 827-833 (2011)CrossRefGoogle Scholar
  9. 9.
    Guerrini, M., Naggi, A., Guglieri, S., Santarsiero, R., Torri, G.: Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy. Anal. Biochem. 337, 35–47 (2005)CrossRefGoogle Scholar
  10. 10.
    Oguma, T., Toyoda, H., Toida, T., Imanari, T.: Analytical method of chondroitin/dermatan sulfates using high performance liquid chromatography/turbo ionspray ionization mass spectrometry: application to analyses of the tumor tissue sections on glass slides. Biomed. Chromatogr. 15, 356–362 (2001)CrossRefGoogle Scholar
  11. 11.
    Zaia, J., Costello, C.E.: Compositional analysis of glycosaminoglycans by electrospray mass spectrometry. Anal. Chem. 73, 233–239 (2000)CrossRefGoogle Scholar
  12. 12.
    Zhang, Z., Linhardt, R.J.: Sequence analysis of native oligosaccharides using negative ESI tandem MS. Curr. Anal. Chem. 5, 225–237 (2009)CrossRefGoogle Scholar
  13. 13.
    Zaia, J., McClellan, J.E., Costello, C.E.: Tandem mass spectrometric determination of the 4s/6s sulfation sequence in chondroitin sulfate oligosaccharides. Anal. Chem. 73, 6030–6039 (2001)CrossRefGoogle Scholar
  14. 14.
    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
  15. 15.
    Zaia, J., Costello, C.E.: Tandem mass spectrometry of sulfated heparin-like glycosaminoglycan oligosaccharides. Anal. Chem. 75, 2445–2455 (2003)CrossRefGoogle Scholar
  16. 16.
    Kailemia, M.J.; Li, L.; Ly, M.; Linhardt, R.J., Amster, I.J.: Complete mass spectral characterization of a synthetic ultralow-molecular-weight heparin using collision-induced dissociation. Anal. Chem. 84, 5475–5478 (2012)CrossRefGoogle Scholar
  17. 17.
    Saad, O.M., Leary, J.A.: Heparin sequencing using enzymatic digestion and ESI-MSn with HOST: a heparin/HS oligosaccharide sequencing tool. Anal. Chem. 77, 5902–5911 (2005)Google Scholar
  18. 18.
    Meissen, J.; Sweeney, M.; Girardi, M.; Lawrence, R.; Esko, J., Leary, J.: Differentiation of 3-O-sulfated heparin disaccharide isomers: identification of structural aspects of the heparin CCL2 binding motif. J. Am. Soc. Mass Spectrom. 20, 652–657 (2009)CrossRefGoogle Scholar
  19. 19.
    Wolff, J.J., Amster, I.J., Chi, L., Linhardt, R.J.: Electron detachment dissociation of glycosaminoglycan tetrasaccharides. J. Am. Soc. Mass Spectrom. 18, 234–244 (2007)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.
    Leach, F.E. III, Wolff, J.J., Laremore, T.N., Linhardt, R.J., Amster, I.J.: Evaluation of the experimental parameters which control electron detachment dissociation, and their effect on the fragmentation efficiency of glycosaminoglycan carbohydrates. Int. J. Mass Spectrom. 276, 110–115 (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.
    Leach, F.E. III, 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
  24. 24.
    Wolff, J.J., Leach, 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
  25. 25.
    Leach, F.E. III, Wolff, J.J., Xiao, Z., Ly, M., Laremore, T.N., Arungundram, S., Al-Mafraji, K., Venot, A., Boons, G.-J., Linhardt, R.J.: Negative electron transfer dissociation Fourier transform mass spectrometry of glycosaminoglycan carbohydrates. Eur. J. Mass Spectrom. 17, 167–176 (2011)CrossRefGoogle Scholar
  26. 26.
    Zaia, J., Miller, M.J.C., Seymour, J.L., Costello, C.E.: The role of mobile protons in negative ion CID of oligosaccharides. J. Am. Soc. Mass Spectrom. 18, 952–960 (2007)CrossRefGoogle Scholar
  27. 27.
    Naggar, E.F., Costello, C.E., Zaia, J.: Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans. J. Am. Soc. Mass Spectrom. 15, 1534–1544 (2004)CrossRefGoogle Scholar
  28. 28.
    Kailemia, M.J., Li, L., Xu, Y., Liu, J., Linhardt, R.J., Amster, I.J.: Structurally informative tandem mass spectrometry of highly sulfated natural and chemoenzymatically synthesized heparin and heparan sulfate glycosaminoglycans. Mol. Cell. Proteom. 12, 979–990 (2013)CrossRefGoogle Scholar
  29. 29.
    Desaire, H., Leary, J.A.: The effects of coordination number and ligand size on the gas phase dissociation and stereochemical differentiation of cobalt-coordinated monosaccharides. Int. J. Mass Spectrom. 209, 171–184 (2001)CrossRefGoogle Scholar
  30. 30.
    Wolff, J.J., Laremore, T.N., Aslam, H., Linhardt, R.J., Amster, I.J.: Electron-induced dissociation of glycosaminoglycan tetrasaccharides. J. Am. Soc. Mass Spectrom. 19, 1449–1458 (2008)CrossRefGoogle Scholar
  31. 31.
    Volpi, N., Maccari, F., Linhardt, R.J.: Capillary electrophoresis of complex natural polysaccharides. Electrophoresis 29, 3095–3106 (2008)CrossRefGoogle Scholar
  32. 32.
    Costell, C., Contado-Miller, J., Cipollo, J.: A glycomics platform for the analysis of permethylated oligosaccharide alditols. J. Am. Soc. Mass Spectrom. 18, 1799–1812 (2007)CrossRefGoogle Scholar
  33. 33.
    Hitchcock, A.M., Costello, C.E., Zaia, J.: Glycoform quantification of chondroitin/dermatan sulfate using a liquid chromatography-tandem mass spectrometry platform. Biochemistry (Mosc) 45, 2350–2361 (2006)Google Scholar
  34. 34.
    Schenauer, M.R., Meissen, J.K., Seo, Y., Ames, J.B., Leary, J.A.: Heparan sulfate separation, sequencing, and isomeric differentiation: ion mobility spectrometry reveals specific iduronic and glucuronic acid-containing hexasaccharides. Anal. Chem. 81, 10179–10185 (2009)CrossRefGoogle Scholar
  35. 35.
    Staples, G.O., Bowman, M.J., Costello, C.E., Hitchcock, A.M., Lau, J.M., Leymarie, N., Miller, C., Naimy, H., Shi, X., Zaia, J.: A chip‐based amide‐HILIC LC/MS platform for glycosaminoglycan glycomics profiling. Proteomics 9, 686–695 (2009)CrossRefGoogle Scholar
  36. 36.
    Bruggink, C., Wuhrer, M., Koeleman, C.A.M., Barreto, V., Liu, Y., Pohl, C., Ingendoh, A., Hokke, C.H., Deelder, A.M.: Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry. J. Chromatogr. B 829, 136–143 (2005)CrossRefGoogle Scholar
  37. 37.
    Maslen, S., Sadowski, P., Adam, A., Lilley, K., Stephens, E.: Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography-MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 78, 8491–8498 (2006)CrossRefGoogle Scholar
  38. 38.
    Jin, L., Barran, P.E., Deakin, J.A., Lyon, M., Uhrín, D.: Conformation of glycosaminoglycans by ion mobility mass spectrometry and molecular modelling. PCCP 7, 3464–3471 (2005)CrossRefGoogle Scholar
  39. 39.
    Fenn, L.S., McLean, J.A.: Structural resolution of carbohydrate positional and structural isomers based on gas-phase ion mobility-mass spectrometry. PCCP 13, 2196–2205 (2011)CrossRefGoogle Scholar
  40. 40.
    Dwivedi, P., Bendiak, B., Clowers, B.H., Hill, H. H. Jr.: Rapid resolution of carbohydrate isomers by electrospray ionization ambient pressure ion mobility spectrometry-time-of-flight mass spectrometry (ESI-APIMS-TOFMS). J. Am. Soc. Mass Spectrom. 18, 1163–1175 (2007)CrossRefGoogle Scholar
  41. 41.
    Clowers, B.H., Dwivedi, P., Steiner, W.E., Hill, J.H.H., Bendiak, B.: Separation of sodiated isobaric disaccharides and trisaccharides using electrospray ionization-atmospheric pressure ion mobility-time of flight mass spectrometry. J. Am. Soc. Mass Spectrom. 16, 660–669 (2005)CrossRefGoogle Scholar
  42. 42.
    Li, H., Giles, K., Bendiak, B., Kaplan, K., Siems, W.F., Hill, H.H. Jr.: Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry. Anal. Chem. 84, 3231–3239 (2012)CrossRefGoogle Scholar
  43. 43.
    Seo, Y., Andaya, A., Leary, J.A.: Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry. Anal. Chem. 84, 2416–2423 (2012)CrossRefGoogle Scholar
  44. 44.
    Schneider, B.B., Covey, T.R., Coy, S.L., Krylov, E.V., Nazarov, E.G.: Chemical effects in the separation process of a differential mobility/mass spectrometer system. Anal. Chem. 82, 1867–1880 (2010)CrossRefGoogle Scholar
  45. 45.
    Purves, R.W., Guevremont, R.: Electrospray ionization high-field asymmetric waveform ion mobility spectrometry-mass spectrometry. Anal. Chem. 71, 2346–2357 (1999)CrossRefGoogle Scholar
  46. 46.
    Barnett, D.A., Ells, B., Guevremont, R., Purves, R.W.: Separation of leucine and isoleucine by electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry. J. Am. Soc. Mass Spectrom. 10, 1279–1284 (1999)CrossRefGoogle Scholar
  47. 47.
    Guevremont, R., Purves, R.W.: High field asymmetric waveform ion mobility spectrometry-mass spectrometry: an investigation of leucine enkephalin ions produced by electrospray ionization. J. Am. Soc. Mass Spectrom. 10, 492–501 (1999)CrossRefGoogle Scholar
  48. 48.
    Gabryelski, W., Froese, K.L.: Rapid and sensitive differentiation of anomers, linkage, and position isomers of disaccharides using high-hield asymmetric waveform ion mobility spectrometry (FAIMS). J. Am. Soc. Mass Spectrom. 14, 265–277 (2003)CrossRefGoogle Scholar
  49. 49.
    Creese, A.J., Cooper, H.J.: Separation and identification of isomeric glycopeptides by high field asymmetric waveform ion mobility spectrometry. Anal. Chem. 84, 2597–2601 (2012)CrossRefGoogle Scholar
  50. 50.
    Robinson, E.W., Garcia, D.E., Leib, R.D., Williams, E.R.: Enhanced mixture analysis of poly(ethylene glycol) using high-field asymmetric waveform ion mobility spectrometry combined with fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 78, 2190–2198 (2006)CrossRefGoogle Scholar
  51. 51.
    Levin, D.S., Vouros, P., Miller, R.A., Nazarov, E.G.: Using a nanoelectrospray-differential mobility spectrometer-mass spectrometer system for the analysis of oligosaccharides with solvent selected control over ESI aggregate ion formation. J. Am. Soc. Mass Spectrom. 18, 502–511 (2007)CrossRefGoogle Scholar
  52. 52.
    Kolakowski, B.M., Mester, Z.: Review of applications of high-field asymmetric waveform ion mobility spectrometry (FAIMS) and differential mobility spectrometry (DMS). Analyst 132, 842–864 (2007)CrossRefGoogle Scholar
  53. 53.
    Shvartsburg, A.A., Bryskiewicz, T., Purves, R.W., Tang, K., Guevremont, R., Smith, R.D.: Field asymmetric waveform ion mobility spectrometry studies of proteins: dipole alignment in ion mobility spectrometry? J. Phys. Chem. B 110, 21966–21980 (2006)Google Scholar
  54. 54.
    Shvartsburg, A.A., Creese, A.J., Smith, R.D., Cooper, H.J.: Separation of peptide isomers with variant modified sites by high-resolution differential ion mobility spectrometry. Anal. Chem. 82, 8327–8334 (2010)CrossRefGoogle Scholar
  55. 55.
    Robinson, E.W., Williams, E.R.: Multidimensional separations of ubiquitin conformers in the gas phase: relating ion cross sections to h/d exchange measurements. J. Am. Soc. Mass Spectrom. 16, 1427–1437 (2005)CrossRefGoogle Scholar
  56. 56.
    Arungundram, S., Al-Mafraji, K., Asong, J., Leach, 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
  57. 57.
    Leach, F. E. III, 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
  58. 58.
    Muñoz, E., Xu, D., Avci, F., Kemp, M., Liu, J., Linhardt, R.J.: Enzymatic synthesis of heparin related polysaccharides on sensor chips: rapid screening of heparin-protein interactions. Biochem. Biophys. Res. Commun. 339, 597–602 (2006)CrossRefGoogle Scholar
  59. 59.
    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
  60. 60.
    Remko, M., von der Lieth, C.-W.: Gas-phase and solution conformations of the α-l-iduronic acid structural unit of heparin. J. Chem. Inf. Model. 46, 1194–1200 (2006)CrossRefGoogle Scholar
  61. 61.
    Mulloy, B., Forster, M.J.: Conformation and dynamics of heparin and heparan sulfate. Glycobiology 10, 1147–1156 (2000)CrossRefGoogle Scholar
  62. 62.
    Pomin, V.H., Sharp, J.S., Li, X., Wang, L., Prestegard, J. H.: Characterization of glycosaminoglycans by 15N NMR spectroscopy and in vivo isotopic labeling. Anal. Chem. 82, 4078–4088 (2010)CrossRefGoogle Scholar
  63. 63.
    Berman, E.S.F., Kulp, K.S., Knize, M.G., Wu, L., Nelson, E.J., Nelson, D.O., Wu, K.J.: Distinguishing monosaccharide stereo- and structural isomers with TOF-SIMS and multivariate statistical analysis. Anal. Chem. 78, 6497–6503 (2006)CrossRefGoogle Scholar
  64. 64.
    Fängmark, I., Jansson, A., Nilsson, B.: Determination of linkage position and anomeric configuration in glucose-containing disaccharide alditols by multivariate analysis of data from mass spectrometry. Anal. Chem. 71, 1105–1110 (1999)CrossRefGoogle Scholar
  65. 65.
    Oh, H., Leach, F., Arungundram, S., Al-Mafraji, K., Venot, A., Boons, G.-J., Amster, I.: 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

Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  • Muchena J. Kailemia
    • 1
  • Melvin Park
    • 2
  • Desmond A. Kaplan
    • 2
  • Andre Venot
    • 3
  • Geert-Jan Boons
    • 3
  • Lingyun Li
    • 4
  • Robert J. Linhardt
    • 4
  • I. Jonathan Amster
    • 1
  1. 1.Department of ChemistryUniversity of GeorgiaAthensUSA
  2. 2.Bruker DaltonicsBillericaUSA
  3. 3.Complex Carbohydrate Research CenterUniversity of GeorgiaAthensUSA
  4. 4.Department of Chemistry and Chemical Biology, Chemical and Biological Engineering, and BiologyRensselaer Polytechnic InstituteTroyUSA

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