, Volume 22, Issue 1–3, pp 17–24 | Cite as

Analysis of recombinat glycoproteins by mass spectrometry

  • David C. James
Special Issue


The advent of new technologies for analysis of biopolymers by mass spectrometry has revolutionised strategies for recombinant protein characterization. The principal recent developments have been matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Using these tools, accurate molecular mass determinations can now be obtained routinely-often using minute (picomole-femtomole) quantities of protein or protein fragments. These techniques have proved indispensible for detailed characterization of the post-translational modifications of recombinant proteins produced by eukaryotic systems. Glycosylation is arguably the most important and complex of these modifications and has prompted widespread use of these new techniques. In this mini-review article I describe recent advances in the use of mass spectrometry for analysis of recombinant glycoproteins.

Key words

mass spectrometry glycosylation recombinant protein oligosaccharide 


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  1. Aebersold R (1993) Mass spectrometry of proteins and peptides in biotechnology. Curr Opin Biotechnol 4: 412–419.Google Scholar
  2. Andersen JS, Svensson B and Roepstorff P (1996) Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: powerful analytical tools in recombinant protein chemistry. Nature Biotechnology 14: 449–457.Google Scholar
  3. Ashton DS, Beddell CR, Cooper DJ, Graig SJ, Lines AC, Oliver RWA and Smith MA (1995) Mass spectrometry of the humanized monoclonal antibody CAMPATH 1H. Anal Chem 67: 835–842.Google Scholar
  4. Bihoreau N, Veillon JF, Ramon C, Scohyers JM and Schmitter JM (1995) Characterization of a recombinant antihaemophilia-A factor (factor-VIII-delta-II) by matrix assisted laser desorption ionization mass spectrometry. Rapid Commun. Mass. Spectrom. 9: 1584–1588.Google Scholar
  5. Billeci TM and Stults JT (1993) Tryptic mapping of recombinant proteins by matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 65: 1709–1716.Google Scholar
  6. Bloom JW, Madanat MS and Ray MK (1996) Cell-line and site specific comparative analysis of the N-linked oligosaccharides on human ICAM/-1DES454–532 by electrospray mass spectrometry. Biochemistry 35: 1856–1864.Google Scholar
  7. Börnsen KO, Mohr MD and Widmer HM (1995) Ion exchange and purification of carbohydrates on a nafion membrane as a new sample pretreatment for matrix-assisted laser desorption/ionization. Rapid Commun Mass Spectrom 9: 1031–1034.Google Scholar
  8. Buchanan MV and Hettich RL (1993) Fourier transform mass spectrometry of high-mass biomolecules. Anal Chem 65: 245A-259A.Google Scholar
  9. Carr SA, Hemling ME, Bean MF and Roberts GD (1991) Integration of mass spectrometry in analytical biotechnology. Anal Chem 63: 2802–2824.Google Scholar
  10. Carr SA, Huddleston MJ and Bean MF (1993) Selective identification and differentiation of N-and O-linked oligosaccharides in glycoproteins by liquid-chromatography-mass spectrometry. Protein Sci 2: 183–196.Google Scholar
  11. Chait BT and Kent SBH (1992) Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. Science 257: 1885–1894.Google Scholar
  12. Clauser KR, Hall SC, Smith DM, Webb JW, Andrews LE, Tran HM, Epstein LB and Burlingame AL (1995) Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE. Proc Natl Acad Sci USA. 92: 5072–5076.Google Scholar
  13. Conboy JJ and Henion JD (1992) The determination of glycopeptides by liquid chromatography/mass spectrometry with collision induced dissociation. J Am Soc Mass Spectrom 3: 804–814.Google Scholar
  14. Dell A, Reason AJ, Khoo K-H, Panico M., McDowell R and Morris H (1994a) Mass spectrometry of carbohydrate-containing biopolymers. Methods Enzymol. 230: 108–132.Google Scholar
  15. Dell A, Khoo KH, Panico M, McDowell RA, Etienne AT, Reason AJ and Morris HR (1994b) FAB-MS and ES-MS of Glycoproteins. In: Fukuda M and Kobata A (eds) Glycobiology: A Practical Approach (p. 187) IRL Press, Oxford.Google Scholar
  16. Dwek RA, Edge CJ, Harvey DJ and Wormald MR (1993) Analysis of glycoprotein-associated oligosaccharides. Annu Rev Biochem 62: 65–100.Google Scholar
  17. Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G and Conradt H (1995) Construction of stable BHK-21-cells co-expressing human secretory glycoproteins and human Gal(β-1–4)GleNAcr α-2,6-sialyltrasferase α-2,6-linked NeuAc is preferentially attached to the Gal(β-1–4)GlcNAc(β-1–2)Man(α-1–3)-branch of diantennary oligosaccharides from secreted recombinant β-trace protein. Eur J Biochem 232: 718–725.Google Scholar
  18. Greis KD, Hayes BK, Comer FI, Kirk M, Barnes S, Lowary TL and Hart GW (1996) Selective identification and site-analysis of OGlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass-spectrometry. Anal Biochem 234: 38–49.Google Scholar
  19. Harvey DJ (1993) Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides. Rapid Commun. Mass Spectrom. 7: 614–619.Google Scholar
  20. Harvey DJ, Rudd PM, Bateman RH, Bordoli RS, Howes K, Hoyes JB and Vickers RG (1994) Examination of complex oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments. Org Mass Spectrom 29: 753–765.Google Scholar
  21. Hemling ME, Mentzer MA, Capiau C and Carr SA (1996) A multifaceted strategy for the characterization of recombinat gD-2, a potential herpes vaccine. In: Burlingame AL, Carr SA (eds) Mass Spectrometry in the Biological Sciences. (pp 307–331) Humana Press, Totowa, New Jersey.Google Scholar
  22. Hooker AD, Goldman MH, Markham NH, James DC, Ison AP, Bull AT, Strange PG, Salmon I, Baines AJ and Jenkins N (1995) N-Glycans of recombinant human interferon-γ change during batch culture of chinese hamster ovary cells. Biotechnol Bioeng 48: 639–648.Google Scholar
  23. Huberty MC, Vath JE, Yu W and Martin SA (1993) Site-specific carbohydrate identification in recombinant proteins using MALD TOF-MS. Anal Chem 65: 2791–2800.Google Scholar
  24. Huddleston MJ, Bean MF and Carr SA (1993) Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and MC/MS/MS: methods for selective detection of glycopeptides in protein digests. Anal Chem 65: 887–884.Google Scholar
  25. Hunter AP and Games DE (1995) Evaluation of glycosylation site heterogeneity and selective identification of glycopeptides in proteolytic digests of bovine α1-acid glycoprotein by mass spectrometry. Rapid Commun Mass Spectrom 9: 42–56.Google Scholar
  26. James DC, Freedman RB, Hoare M, Ogpnah OW, Rooney BC, Larinov OA, Dobrolvolsky VN, Lagutin OV and Jenkins N (1995) N-Glycosylation of recombinant human interferon-γ produced in animal expression systems. Bio/Technology 13: 592–596.Google Scholar
  27. James DC, Goldman MH, Hoare M, Jenkins N, Oliver RWA, Green BN and Freedman RB (1996) Posttranslational processing of recombinant human interferon-γ in animal expression systems. Protein Sci 5: 331–340.Google Scholar
  28. Jenkins N and Curling EM (1994) Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol 16: 354–364.Google Scholar
  29. Karas M, Bahr U, Strupat K, Hillenkamp F, Tsarbopoulos A and Pramanik BN (1995) Matrix dependence of metastable fragmentation of glycoproteins in MALDI TOF mass spectrometry. Anal Chem 67: 675–679.Google Scholar
  30. Kelly JF, Locke SJ, Ramaley L and Thibault P (1996) Development of electrophoretic conditions for the characterization of protein glycoforms by capillary electrophoresis electrospray mass spectrometry. J Chromatog 720: 409–427.Google Scholar
  31. Kragten EA, Bergwerff AA, van Oostrum J, Müller DL and Richter WJ (1995) Site-specific analysis of the N-glycans on mirine polymeric immunoglobulin-A using liquid chromatography electrospray mass spectrometry. J. Mass Spectrom 30: 1679–1686.Google Scholar
  32. Lifely MR, Hale C, Boyce S, Keen MJ, and Phillips J (1995) Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions. Glycobiology 5: 813–822.Google Scholar
  33. Ling V, Guzzetta AW, Canova-Davis E, Stults JT, Hancock WS, Covey TR and Shushan BI (1991) Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activatorby high performance liquid chromatography-electrospray ionization mass spectrometry. Anal Chem 63: 2909–2915.Google Scholar
  34. Linsley KB, Chan SY, Chan S, Reinhold BB, Lisi PJ and Reinhold VN (1994) Applications of electrospray mass-spectrometry to erythropoietin N-linked and O-linked glycans. Anal Biochem 219: 207–217.Google Scholar
  35. Liu DTY (1992) Glycoprotein pharmaceuticals—scientific and regulatory considerations, and the United-States Orphan Drug-Act. Trends Biotechnol 10: 114–120.Google Scholar
  36. Mann M and Wilm M (1995) Electrospray mass spectrometry for protein characterization. TIBS 20: 219–224.Google Scholar
  37. Medzihradszky KF, Maltby DA, Hall SC, Settineri CA and Burlingame AL (1994) Characterization of protein N-glycosylation by reversed-phase microbore liquid chromatography electrospray mass spectrometry, complimentary mobile phases and sequential exoglycosidase digestion. J Am Soc Mass Spectrom 5: 350–358.Google Scholar
  38. Mock KK, Davey M and Cottrell JS (1991) The analysis of underivatised oligosaccharides by matrix-assisted laser desoption massspectrometry. Biochem Biophys Res Commun 177: 644–651.Google Scholar
  39. Mock KK, Sutton CW and Cottrell JS (1992) Sample immobilization protocols for matrix-assisted laser desorption mass spectrometry. Rapid Commun. Mass Spectrom. 6: 233–238.Google Scholar
  40. Mohr MD, Börnsen KO and Widmer HM (1995) Matrix-assisted laser desorption ionization mass spectrometry-improved matrix for oligosaccharides. Rapid Commun Mass Spectrom 9: 809–814.Google Scholar
  41. Müller D, Domon B, Karas M, van Oostrum J and Richter WJ (1994) Characterization and direct glycoform profiling of a hybrid plasminogen activator by matrix-assisted laser desorption and electrospray mass spectrometry: correlation with high-performance liquid chromatographic and nuclear magnetic resonance analyses of the released glycans. Biol Mass Spectrom 23: 330–338.Google Scholar
  42. Nguyen DN, Becker GW and Riggin RM (1995) Protein mass spectrometry-applications to analytical biotechnology. J Chromatog 705: 21–45.Google Scholar
  43. Ogonah OW, Freedman RB, Jenkins N, Patel K and Rooney BC (1996) Isolation and characterization of an insect cell line able to perform complex N-linked glycosylation of recombinant proteins. Bio/Technology 14: 197–202.Google Scholar
  44. Peter-Katalinic J (1994) Analysis of glycoconjugates by fast atom bombardment mass spectrometry and related MS techniques. Mass Spectrom Revs 13: 77–98.Google Scholar
  45. Roberts GD, Johnson WP, Burman S, Anumula KR and Carr SA (1995) An integrated strategy for structural characterization of the protein and carbohydrate components of monoclonal antibodies: application to anti-respiratory syncytial virus mAb. Anal Chem 67: 3613–3625.Google Scholar
  46. Robinson DK, Chan CP, Up VY, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J and Silberklang M (1994) Characterization of a recombinant antibody produced in the course of a high-yield fed-batch process. Biotechnol Bioeng 44: 727–735.Google Scholar
  47. Rush RS, Derby PL, Smith DM, Merry C, Rogers G, Rohde MF and Katta V (1995) Microheterogeneity of erythropoietin carbohydrate structure. Anal Chem 67: 1442–1452.Google Scholar
  48. Schindler PA, Settineri CA, Collet X, Fielding CJ and Burlingame AL (1995) Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion. Protein Sci 4: 791–801.Google Scholar
  49. Siuzdak G (1994) The emergence of mass spectrometry in biochemical research. Proc Natl Acad Sci USA 91: 11290–11297.Google Scholar
  50. Smith RD and Light-Wahl KJ (1993) The observation of noncovalent interactions in solution by electrospray ionization mass spectrometry: promise, pitfalls and prognosis. Biol Mass Spectrom 22: 493–501.Google Scholar
  51. Stahl B, Steup M, Karas M and Hillenkamp F (1991) Analysis of neutral oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 63: 1463–1466.Google Scholar
  52. Stone KL, LoPresti MB, Crawford JM, De Angelis R and Williams KR (1989) Enzymatic Digestion of Proteins and HPLC Peptide Isolation. In: Matsudaira P (ed.) A Practical Guide to Protein and Peptide Purification for Microsequencing. (p. 31) Academic Press, San Diego-London.Google Scholar
  53. Stults JT (1995) Matrix-assisted laser desorption/ionization mass spectrometry. Curr Opin Struct Biol 5: 691–698.Google Scholar
  54. Sutton CW, O'Neill J and Cottrell JS (1994) Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. Anal Biochem 218: 34–46.Google Scholar
  55. Taguchi T, Seko A, Kitajima K, Muto Y, Inoue S, Khoo KH, Morris HR, Dell A and Inoue Y (1994) Structural studies of a novel type of pentaantennary large glycan unit in the fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of Oryzias latipes. J Biol Chem 269: 8762–8771.Google Scholar
  56. Treuheit MJ, Costello CE and Halsall HB (1992) Analysis of the five glycosylation sites of human α1-acid glycoprotein. Biochem J 283: 105–112.Google Scholar
  57. Tsarbopoulos A, Karas M, Strupat K, Pramanik BN, Nagabhushan TL and Hillenkamp F (1994) Comparative mapping of recombinant proteins and glycoproteins by plasma desorption and matrix-assisted laser desorption mass spectrometry. Anal Chem 66: 2062–2070.Google Scholar
  58. Tsarbopoulos A, Pramanik BN, Nagabhushan TL and Covey TR (1995) Structural analysis of the CHO-derived interleukin-4 by liquid chromatography electrospray ionization mass spectrometry. J Mass Spectom 30: 1752–1763.Google Scholar
  59. Vestal ML, Juhasz P and Martin SA (1995) Delayed extraction matrix-assisted laser desorption time-of-flight mass spectrometry. Rapid Commun Mass Spectron 9: 1044–1050.Google Scholar
  60. Wilm M and Mann M (1996) Analytical properties of the nanoelectrospray ion-source. Anal Chem 68: 1–8.Google Scholar
  61. Yu Ip CC Miller WJ, Silberklang M, Mark GE, Ellis RQ, Huang L, Glushka J, VanHalbeek H, Zhu J and Alhadeff JA (1994) Structural characterization of the N-glycans of a humanized anti-CD18 murine immunoglobulin G. Arch Biochem Biophys 308: 387–399.Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • David C. James
    • 1
  1. 1.Research School of BiosciencesUniversity of KentCanterburyUK

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