N-Linked Global Glycan Profiling by NanoLC Mass Spectrometry

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 790)

Abstract

A method is detailed for the global profiling of underivatized N-linked glycans that are derived from complex protein mixtures. The method consists of five main steps that include the following: (1) protein denaturation; (2) enzymatic digestion; (3) solid phase extraction; (4) nanoLC MS analysis; and (5) data interpretation. Materials, methods, and algorithms for the identification of both glycan composition and structure are summarized. In addition, potential problems and their resolutions are addressed.

Key words

N-linked glycans NanoLC mass spectrometry Glycoproteins Glycosylation Glycan profiling Hydrophilic interaction chromatography Glycobiology Glycan algorithms 

References

  1. 1.
    Blow, N. (2009) Glycobiology: A spoonful of sugar. Nature 457, 617–620.PubMedCrossRefGoogle Scholar
  2. 2.
    Wu, H.C., Meezan, E., Black, P.H., et al. (1969) Comparative Studies on Carbohydrate-Containing Membrane Components of Normal and Virus-Transformed Mouse Fibroblasts.I. Glucosamine-Labeling Patterns in 3 t3 Spontaneously Transformed 3 t3 and Sv-40-Transformed 3 t3 Cells. Biochemistry 8, 2509–2517.PubMedCrossRefGoogle Scholar
  3. 3.
    Bereman, M.S., Williams, T.I., Muddiman, D.C. (2009) Development of a nanoLC LTQ Orbitrap Mass Spectrometric Method for Profiling Glycans Derived from Plasma from Healthy, Benign Tumor Control, and Epithelial Ovarian Cancer Patients. Anal Chem 81, 1130–1136.PubMedCrossRefGoogle Scholar
  4. 4.
    Li, B., An, H.J., Kirmiz, C., et al. (2008) Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins. J Proteome Res 7, 3776–3788.PubMedCrossRefGoogle Scholar
  5. 5.
    Kirmiz, C., Li, B.S., An, H.J., et al. (2007) A serum glycomics approach to breast cancer biomarkers, Mol Cell Proteomics 6, 43–55.PubMedGoogle Scholar
  6. 6.
    An, H.J., Miyamoto, S., Lancaster, K.S., et al. (2006) Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer. J Proteome Res 5, 1626–1635.PubMedCrossRefGoogle Scholar
  7. 7.
    Mechref, Y., Hussein, A., Bekesova, S., et al. (2009) Quantitative Serum Glycomics of Esophageal Adenocarcinoma and Other Esophageal Disease Onsets. J Proteome Res 8, 2656–2666.PubMedCrossRefGoogle Scholar
  8. 8.
    Isailovic, D., Kurulugama, R.T., Plasencia, M.D., et al. (2008) Profiling of human serum glycans associated with liver cancer and cirrhosis by IMS-MS. J Proteome Res 7, 1109–1117.PubMedCrossRefGoogle Scholar
  9. 9.
    Kyselova, Z., Mechref, Y., Kang, P., et al. (2008) Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles. Clin Chem 54, 1166–1175.PubMedCrossRefGoogle Scholar
  10. 10.
    Kyselova, Z., Mechref, Y., Al Bataineh, M.M., et al. (2007) Alterations in the serum glycome due to metastatic prostate cancer. J Proteome Res 6, 1822–1832.PubMedCrossRefGoogle Scholar
  11. 11.
    Harvey, D.J. (1999) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom Rev 18, 349–450.PubMedCrossRefGoogle Scholar
  12. 12.
    Karlsson, N.G., Wilson, N.L., Wirth, H.J., et al. (2004) Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. Rapid Commun Mass Spectrom 18, 2282–2292.PubMedCrossRefGoogle Scholar
  13. 13.
    Barroso, B., Dijkstra, R., Geerts, M., et al. (2002) On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins. Rapid Commun Mass Spectrom 16, 1320–1329.PubMedCrossRefGoogle Scholar
  14. 14.
    Bahr, U., Pfenninger, A., Karas, M., et al. (1997) High sensitivity analysis of neutral underivatized oligosaccharides by nanoelectrospray mass spectrometry. Anal Chem 69, 4530–4535.PubMedCrossRefGoogle Scholar
  15. 15.
    Karas, M., Bahr, U., Dulcks, T. (2000) Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine. Fresenius J Anal Chem 366, 669–676.PubMedCrossRefGoogle Scholar
  16. 16.
    Ciucanu, I., Kerek, F. (1984) A Simple and Rapid Method for the Permethylation of Carbohydrates. Carbohydr Res 131, 209–217.CrossRefGoogle Scholar
  17. 17.
    Dell, A. (1990) Preparation and Desorption Mass-Spectrometry of Permethyl and Peracetyl Derivatives of Oligosaccharides. Methods Enzymol 193, 647–660.PubMedCrossRefGoogle Scholar
  18. 18.
    Pabst, M., Bondili, J.S., Stadlmann, J., et al. (2007) Mass plus 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.PubMedCrossRefGoogle Scholar
  19. 19.
    Tretter, V., Altmann, F., März, L. (1991) Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1–3 to the asparagine-linked N-acetylglucosamine residue. Eur J Biochem 199, 647–652.PubMedCrossRefGoogle Scholar
  20. 20.
    Kuster, B., Harvey, D.J. (1997) Ammonium containing buffers should be avoided during enzymatic release of glycans from glycoproteins when followed by reducing terminal derivatization. Glycobiology 7, vii-ix.Google Scholar
  21. 21.
    Bereman, M.S., Young, D.D., Deiters, A., et al. (2009) Development of a robust and high throughput method for profiling N-linked glycans derived from plasma glycoproteins by NanoLC-FTICR mass spectrometry. J Proteome Res 8, 3764–3770.PubMedCrossRefGoogle Scholar
  22. 22.
    Tarentino, A.L., Plummer, T.H., Jr. (1994) Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol 230, 44–57.PubMedCrossRefGoogle Scholar
  23. 23.
    Plummer, T.H., Jr., Elder, J.H., Alexander, S., et al. (1984) Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations. J Biol Chem 259, 10700–10704.PubMedGoogle Scholar
  24. 24.
    Tarentino, A.L., Gomez, C.M., Plummer, T.H. (1985) Deglycosylation of Asparagine-Linked Glycans by Peptide - N-Glycosidase-F. Biochemistry 24, 4665–4671.PubMedCrossRefGoogle Scholar
  25. 25.
    Williams, T.I., Saggese, D.A., Toups, K.L., et al. (2008) Investigations with O-linked protein Glycosylations by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J Mass Spectrom 43, 1215–1223.PubMedCrossRefGoogle Scholar
  26. 26.
    Davies, M.J., Smith, K.D., Carruthers, R.A., et al. (1993) Use of a Porous Graphitized Carbon Column for the High-Performance Liquid-Chromatography of Oligosaccharides, Alditols and Glycopeptides with Subsequent Mass-Spectrometry Analysis. J Chromatogr 646, 317–326.PubMedCrossRefGoogle Scholar
  27. 27.
    Alpert, A.J. (1990) Hydrophilic-Interaction Chromatography for the Separation of Peptides, Nucleic-Acids and Other Polar Compounds. J Chromatogr 499, 177–196.PubMedCrossRefGoogle Scholar
  28. 28.
    Pabst, M., Altmann, F. (2008) 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.PubMedCrossRefGoogle Scholar
  29. 29.
    Bereman, M.S., Muddiman, D.C. (2010) The effects of abundant plasma protein depletion on global glycan profiling using nanoLC FT-ICR mass spectrometry. Anal Bioanal Chem 396, 1473–1479.PubMedCrossRefGoogle Scholar
  30. 30.
    Ceroni, A., Maass, K., Geyer, H., et al. (2008) GlycoWorkbench: A tool for the computer-assisted annotation of mass spectra of Glycans. J Proteome Res 7, 1650–1659.PubMedCrossRefGoogle Scholar
  31. 31.
    Maass, K., Ranzinger, R., Geyer, H., et al. (2007) “Glyco-peakfinder” – de novo composition analysis of glycoconjugates. Proteomics 7, 4435–4444.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Genome SciencesUniversity of WashingtonSeattleUSA
  2. 2.Department of ChemistryNorth Carolina State UniversityRaleighUSA

Personalised recommendations