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Ion mobility-mass correlation trend line separation of glycoprotein digests without deglycosylation

  • Original Research
  • Published:
International Journal for Ion Mobility Spectrometry

Abstract

A high-throughput ion mobility mass spectrometer (IMMS) was used to rapidly separate and analyze peptides and glycopeptides derived from glycoproteins. Two glycoproteins, human α-1-acid glycoprotein and antithrombin III were digested with trypsin and subjected to electro-spray traveling wave IMMS analysis. No deglycosylation steps were performed; samples were complex mixtures of peptides and glycopeptides. Peptides and glycosylated peptides with different charge states (up to 4 charges) were observed and fell on distinguishable trend lines in 2-D IMMS spectra in both positive and negative modes. The trend line separation patterns matched between both modes. Peptide sequence was identified based on the corresponding extracted mass spectra and collision induced dissociation (CID) experiments were performed for selected compounds to prove class identification. The signal-to-noise ratio of the glycopeptides was increased dramatically with ion mobility trend line separation compared to non-trend line separation, primarily due to selection of precursor ion subsets within specific mobility windows. In addition, isomeric mobility peaks were detected for specific glycopeptides. IMMS demonstrated unique capabilities and advantages for investigating and separating glycoprotein digests in this study and suggests a novel strategy for rapid glycoproteomics studies in the future.

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References

  1. Eiceman GA, Karpas Z (2005) Ion mobility spectrometry, 2nd edn. Taylor and Francis Group, LLC, Boca Raton

    Book  Google Scholar 

  2. Eiceman GA (2002) Ion-mobility spectrometry as a fast monitor of chemical composition. Trends Anal Chem 21:259–275

    Article  CAS  Google Scholar 

  3. Ewing RG, Atkinson DA, Eiceman GA, Ewing GJ (2001) A critical review of ion mobility spectrometry for the detection of explosives and explosive related compounds. Talanta 54:515–529

    Article  CAS  Google Scholar 

  4. Kanu AB, Hill HH Jr (2007) Identity confirmation of drugs and explosives in ion mobility spectrometry using a secondary drift gas. Talanta 73:692–699

    Article  CAS  Google Scholar 

  5. Kanu AB, Dwivedi P, Tam M, Matz L, Hill HH Jr (2008) Ion mobility-mass spectrometry. J Mass Spectrom 43:1–22

    Article  CAS  Google Scholar 

  6. Dwivedi P, Schultz AJ, Hill HH Jr (2010) Metabolic profiling of human blood by high-resolution ion mobility mass spectrometry (IM-MS). Int J Mass Spectrom 298:78–90

    Article  CAS  Google Scholar 

  7. Kaplan K, Dwivedi P, Davidson S, Yang Q, Tso P, Siems W, Hill HH Jr (2009) Monitoring dynamic changes in lymph metabolome of fasting and fed rats by electrospray ionization-ion mobility mass spectrometry (ESI-IMMS). Anal Chem 81:7944–7953

    Article  CAS  Google Scholar 

  8. Clowers BH, Dwivedi P, Steiner WE, Hill HH Jr (2005) 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

    Article  CAS  Google Scholar 

  9. Isailovic D, Kurulugama RT, Plasencia MD, Stokes ST, Kyselova Z, Goldman R, Mechref Y, Novotny MV, Clemmer DE (2008) Profiling of human serum glycans associated with liver cancer and cirrhosis by IMS-MS. J Proteome Res 7:1109–1117

    Article  CAS  Google Scholar 

  10. Zhu M, Bendiak B, Clowers BH, Hill HH Jr (2009) Ion mobility mass spectrometry analysis of isomeric carbohydrate precursor ions. Anal Bioanal Chem 394:1853–1867

    Article  CAS  Google Scholar 

  11. Eiceman GA, Young D, Smith GB (2005) Mobility spectrometry of amino acids and peptides with matrix assisted laser desorption and ionization in air at ambient pressure. Microchem J 81:108–116

    Article  CAS  Google Scholar 

  12. Valentine SJ, Plasencia MD, Liu X, Krishnan M, Naylor S, Udseth HR, Smith RD, Clemmer DE (2006) Toward plasma proteome profiling with ion mobility-mass spectrometry. J Proteome Res 5:2977–2984

    Article  CAS  Google Scholar 

  13. Wu C, Siems WF, Klasmeier J, Hill HH Jr (2000) Separation of isomeric peptides using electrospray ionization/high-resolution ion mobility spectrometry. Anal Chem 72:391–395

    Article  CAS  Google Scholar 

  14. Fenn LS, Kliman M, Mahsut A, Zhao SR, Mclean JA (2009) Characterizing ion mobility- mass spectrometry conformation space for the analysis of complex biological samples. Anal Bioanal Chem 394:235–244

    Article  CAS  Google Scholar 

  15. Plasencia ND, Isailovic D, Merenbloom SI, Mechref Y, Clemmer DE (2008) Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-MS. J Am Soc Mass Spectrom 19:1706–1715

    Article  CAS  Google Scholar 

  16. Williams JP, Bugarcic T, Habtemariam A, Giles K, Campuzano I, Rodger PM, Sadler PJ (2009) Isomer separation and gas-phase configurations of organoruthenium anticancer complexes: ion mobility mass spectrometry and modeling. J Am Soc Mass Spectrom 20:1119–1122

    Article  CAS  Google Scholar 

  17. Morris HR, Chalabi S, Panico M, Sutton-Smith M, Clark GF, Goldberg D, Dell A (2007) Glycoproteomics: past, present and future. Int J Mass Spectrom 259:16–31

    Article  CAS  Google Scholar 

  18. An HJ, Kronewitter SR, de Leoz MLA, Lebrilla CB (2009) Glycomics and disease markers. Curr Opin Chem Biol 13:601–607

    Article  CAS  Google Scholar 

  19. Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA (2001) Glycosylation and the immune system. Science 291:2370–2376

    Article  CAS  Google Scholar 

  20. Morelle W, Michalski JC (2005) Glycomics and mass spectrometry. Curr Pharm Des 11:2615–2645

    Article  CAS  Google Scholar 

  21. Imre T, Schlosser G, Pocsfalvi G, Siciliano R, Szöllösi É, Kremmer T, Malorni A, Vékey K (2005) Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry. J Mass Spectrom 40:1472–1483

    Article  CAS  Google Scholar 

  22. Kremmer T, Szöllösi É, Boldizsár M, Vincze B, Ludányi K, Imre T, Schlosser G, Vékey K (2004) Liquid chromatographic and mass spectrometric analysis of human serum acid alpha-1-glycoprotein. Biomed Chromatogr 18:323–329

    Article  CAS  Google Scholar 

  23. Fenn LS, McLean JA (2009) Simultaneous glycoproteomics on the basis of structure using ion mobility-mass spectrometry. Mol Biosyst 5:1298–1302

    Article  CAS  Google Scholar 

  24. Olivova P, Chen W, Chakraborty AB, Gebler JC (2008) Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 22:29–40

    Article  CAS  Google Scholar 

  25. Giles K, Pringle SD, Worthington KR, Little D, Wildgoose JL, Bateman RH (2004) Applications of a travelling wave-based radio-frequency only stacked ring ion guide. Rapid Commun Mass Spectrom 18:2401–2414

    Article  CAS  Google Scholar 

  26. Pringle SD, Giles K, Wildgoose JL, Williams JP, Slade SE, Thalassinos K, Bateman RH, Bowers MT, Scrivens JH (2007) An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom 261:1–12

    Article  CAS  Google Scholar 

  27. Alley WR Jr, Mechref Y, Novotny MV (2009) Characterization of glycopeptides by combining collision-induced dissociation and electron-transfer dissociation mass spectrometry data. Rapid Commun Mass Spectrom 23:161–170

    Article  CAS  Google Scholar 

  28. Bongers J, Devincentis J, Fu J, Huang P, Kirkley DH, Leister K, Liu P, Ludwig R, Rumney K, Tao L, Wu W, Russell RJ (2011) Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping. J Chromatog A 1218:8140–8149

    Article  CAS  Google Scholar 

  29. Hanisch FG (2012) O-glycoproteinomics: site-specific O-glycoprotein analysis by CID/ETD electrospray ionization tandem mass spectrometry and top-down glycoprotein sequencing by in-source decay MALDI mass spectrometry. Meth Mol Biol 842:179–189

    Article  CAS  Google Scholar 

  30. Wang D, Hincapie M, Rejtar T, Karger BL (2011) Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular (PLOT) LC-LTQ-CID/ETD-MS. Anal Chem 83:2029–2037

    Article  CAS  Google Scholar 

  31. Giles K, Wildgoose JL, Langridge DJ, Campuzano I (2010) A method for direct measurement of ion mobilities using a travelling wave ion guide. Int J Mass Spectrom 298:10–16

    Article  CAS  Google Scholar 

  32. Shvartsburg AA, Smith RD (2008) Fundamentals of traveling wave ion mobility spectrometry. Anal Chem 80:9689–9699

    Article  CAS  Google Scholar 

  33. Fournier T, Medjoubi-N N, Porquet D (2000) Alpha-1-acid glycoprotein. Biochim Biophys Acta 1482:157–171

    Article  CAS  Google Scholar 

  34. Fournet B, Montreuil J, Strecker G, Dorland L, Haverkamp J, Vliegenthart JFG, Binette JP, Schmid K (1978) Determination of the primary structures of 16 asialo-carbohydrate units derived from human plasma alpha 1-acid glycoprotein by 360-MHz 1H NMR spectroscopy and permethylation analysis. Biochemistry 17:5206–5214

    Article  CAS  Google Scholar 

  35. Treuheit MJ, Costello CE, Halsall HB (1992) Analysis of the five glycosylation sites of human α1-acid glycoprotein. Biochem J 283:105–112

    CAS  Google Scholar 

  36. Cardon P, Parente JP, Leroy Y, Montreuil J, Fournet B (1986) Separation of sialyl-oligosaccharides by high-performance liquid chromatography: application to the analysis of mono-, di-, tri- and tetrasialyl-oligosaccharides obtained by hydrazinolysis of α1-acid glycoprotein. J Chromatogr 356:135–146

    Article  CAS  Google Scholar 

  37. van Halbeek H, Dorland L, Vliegenthart JFG, Montreuil J, Fournet B, Schmid K (1981) Characterization of the microheterogeneity in glycoproteins by 500-MHz 1H NMR spectroscopy of glycopeptide preparations. J Biol Chem 256:5588–5590

    Google Scholar 

  38. Spiro RG (1966) Analysis of sugars found in glycoproteins. Meth Enzymol 8:3–26

    Article  CAS  Google Scholar 

  39. Bunkenborg J, Pilch BJ, Podtelejnikov AV, Wiśniewski JR (2004) Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics 4:454–465

    Article  CAS  Google Scholar 

  40. Picard V, Ersdal-Badju E, Bock SC (1995) Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the β-antithrombin III isoform with enhanced heparin affinity. Biochemistry 34:8433–8440

    Article  CAS  Google Scholar 

  41. McCoy AJ, Pei XY, Skinner R, Abrahams JP, Carrell RW (2003) Structure of β-antithrombin and the effect of glycosylation on antithrombin’s heparin affinity and activity. J Mol Biol 326:823–833

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the National Institutes of Health with grant # 5R33RR020046.

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Authors and Affiliations

  1. Department of Chemistry, Washington State University, Pullman, WA, USA

    Hongli Li, William F. Siems & Herbert H. Hill Jr.

  2. Department of Cell and Developmental Biology, Program in Structural Biology and Biophysics, University of Colorado, Health Sciences Center, Anschutz Medical Campus, Aurora, CO, USA

    Brad Bendiak

  3. Institute of Biological Chemistry, Washington State University, Pullman, WA, USA

    David R. Gang

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  1. Hongli Li
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  2. Brad Bendiak
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  3. William F. Siems
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Correspondence to Herbert H. Hill Jr..

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Li, H., Bendiak, B., Siems, W.F. et al. Ion mobility-mass correlation trend line separation of glycoprotein digests without deglycosylation. Int. J. Ion Mobil. Spec. 16, 105–115 (2013). https://doi.org/10.1007/s12127-013-0127-3

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  • DOI: https://doi.org/10.1007/s12127-013-0127-3

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