Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

  • Jason J. Cournoyer
  • Cheng Lin
  • Michael J. Bowman
  • Peter B. O’Connor
Articles

Abstract

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, zn-57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments.

References

  1. 1.
    Robinson, A. B. Robinson, N. E. Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins; Althouse Press: Cave Junction, OR, 2004.Google Scholar
  2. 2.
    Clarke, S. Propensity for Spontaneous Succinimide Formation from Aspartyl and Asparaginyl Residues in Cellular Proteins. Int. J. Pept. Protein Res. 1987, 30, 808–821.CrossRefGoogle Scholar
  3. 3.
    Aswad, D. W. Paranandi, M. V. Schurter, B. T. Isoaspartate in Peptides and Proteins: Formation, Significance, and Analysis. J. Pharm. Biomed. Anal. 2000, 21, 1129–1136.CrossRefGoogle Scholar
  4. 4.
    Hanson, S. R. A. Hasan, A. Smith, D. L. Smith, J. B. The Major in Vivo Modifications of the Human Water-Insoluble lens Crystallins are Disulfide Bonds, Deamidation, Methionine Oxidation, and Backbone Cleavage. Exp. Eye Res. 2000, 71, 195–207.CrossRefGoogle Scholar
  5. 5.
    Nilsson, M. R. Driscoll, M. Raleigh, D. P. Low Levels of Asparagine Deamidation Can Have a Dramatic Effect on Aggregation of Amyloidogenic Peptides: Implications for the Study of Amyloid Formation. Protein Sci. 2002, 11, 342–349.CrossRefGoogle Scholar
  6. 6.
    Ribot, W. J. Powell, B. S. Ivins, B. E. Little, S. F. Johnson, W. M. Hoover, T. A. Norris, S. L. Adamovicz, J. J. Friedlander, A. M. Andrews, G. P. Comparative Vaccine Efficacy of Different Isoforms of Recombinant Protective Antigen Against Bacillus anthracis Spore Challenge in Rabbits. Vaccine 2006, 24, 3469–3476.CrossRefGoogle Scholar
  7. 7.
    Gershon, H. Gershon, D. Inactive Enzyme Molecules in Aging Mice: Liver Aldolase. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 909–913.CrossRefGoogle Scholar
  8. 8.
    Deverman, B. E. Cook, B. L. Manson, S. R. Niederhoff, R. A. Langer, E. M. Rosova, I. Kulans, L. A. Fu, X. Y. Weinberg, J. S. Heinecke, J. W. Roth, K. A. Weintraub, S. J. Bcl-X-L Deamidation is a Critical Switch in the Regulation of the Response to DNA Damage. Cell 2002, 111, 51–62.CrossRefGoogle Scholar
  9. 9.
    Friedman, A. R. Ichhpurani, A. K. Brown, D. M. Hillman, R. M. Krabill, L. F. Martin, R. A. Zurcherneely, H. A. Guido, D. M. Degradation of Growth-Hormone Releasing-Factor Analogs in Neutral Aqueous-Solution Is Related to Deamidation of Asparagine Residues—Replacement of Asparagine Residues by Serine Stabilizes. Int. J. Pept. Protein Res. 1991, 37, 14–20.CrossRefGoogle Scholar
  10. 10.
    Robinson, N. E. Robinson, A. B. Molecular Clocks. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 944–949.CrossRefGoogle Scholar
  11. 11.
    Robinson, N. E. Robinson, A. B. Prediction of Protein Deamidation Rates from Primary and Three-Dimensional Structure. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 4367–4372.CrossRefGoogle Scholar
  12. 12.
    Hellman, U. Wernstedt, C. Gonez, J. Heldin, C. H. Improvement of an In-Gel Digestion Procedure for the Micropreparation of Internal Protein-Fragments for Amino-Acid Sequencing. Anal. Biochem. 1995, 224, 451–455.CrossRefGoogle Scholar
  13. 13.
    Krokhin, O. V., Antonovici, M., Ens, W., Wilkins, J. A., Standing, K. G. Deamidation of -Asn-Gly- Sequences during Sample Preparation for Proteomics: Consequences for MALDI and HPLC-MALDI Analysis. Anal. Chem. 2006, Web Release July 21, 2006.Google Scholar
  14. 14.
    Hsu, Y. R. Chang, W. C. Mendiaz, E. A. Hara, S. Chow, D. T. Mann, M. B. Langley, K. E. Lu, H. S. Selective Deamidation of Recombinant Human Stem Cell Factor During in Vitro Aging; Isolation and Characterization of the Aspartyl and Isoaspartyl Homodimers and Heterodimers. Biochemistry 1998, 37, 2251–2262.CrossRefGoogle Scholar
  15. 15.
    Geiger, T. Clarke, S. Deamidation, Isomerization, and Racemization at Asparaginyl and Aspartyl Residues in Peptides. J. Biol. Chem. 1987, 262, 785–795.Google Scholar
  16. 16.
    Potter, S. M. Henzel, W. J. Aswad, D. W. In Vitro Aging of Calmodulin Generates Isoaspartate at Multiple Asn-Gly and Asp-Gly Sites in Calcium-Binding Domain-Ii, Domain-Iii, and Domain-Iv. Protein Sci. 1993, 2, 1648–1663.CrossRefGoogle Scholar
  17. 17.
    Capasso, S. Thermodynamic Parameters of the Reversible Isomerization of Aspartic Residues via a Succinimide Derivative. Thermochim. Acta 1996, 286, 41–50.CrossRefGoogle Scholar
  18. 18.
    Meinwald, Y. C. Stimson, E. R. Scheraga, H. A. Deamidation of the Asparaginyl-Glycyl Sequence. Int. J. Pept. Protein Res. 1986, 28, 79–84.CrossRefGoogle Scholar
  19. 19.
    Chelius, D. Rehder, D. S. Bondarenko, P. V. Identification and Characterization of Deamidation Sites in the Conserved Regions of Human Immunoglobulin γ Anitbodies. Anal. Chem. 2005, 77, 6004–6011.CrossRefGoogle Scholar
  20. 20.
    Cournoyer, J. J. Lin, C. O’Connor, P. B. Detecting Deamidation Products in Proteins by Electron Capture Dissociation. Anal. Chem. 2006, 78, 1264–1271.CrossRefGoogle Scholar
  21. 21.
    Tyler-Cross, R. Schirch, V. Effects of Amino Acid Sequence, Buffers, and Ionic Strength on the Rate and Mechanism of Deamidation of Asparagine Residues in Small Peptides. J. Biol. Chem. 1991, 266, 22549–22556.Google Scholar
  22. 22.
    Athiner, L. Kindrachuk, J. Georges, F. Napper, S. The Influence of Protein Structure on the Products Emerging from Succinimide Hydrolysis. J. Biol. Chem. 2002, 277, 30502–30507.CrossRefGoogle Scholar
  23. 23.
    Capasso, S. Di Cerbo, P. Kinetic and Thermodynamic Control of the Relative Yield of Deamidation of Asparagine and Isomerization of Aspartic Acid Residues. J. Peptide Res. 2000, 56, 382–387.CrossRefGoogle Scholar
  24. 24.
    Cirrito, T. P. Pu, Z. Deck, M. B. Unanue, E. R. Deamidation of Asparagine in a Major Histocompatibility Complex-Bound Peptide Affects T Cell Recognition but Does Not Explain Type B Reactivity. J. Exp. Med. 2001, 194, 1165–1170.CrossRefGoogle Scholar
  25. 25.
    Carslon, A. D. Riggin, R. M. Development of Improved High-Performance Liquid Chromatography Conditions for Nonisotopic Detection of Isoaspartic Acid to Determine the Extent of Protein Deamidation. Anal. Biochem. 2000, 278, 150–155.CrossRefGoogle Scholar
  26. 26.
    Huang, L. H. Li, J. R. Wroblewski, V. J. Beals, J. M. Riggin, R. M. In Vivo Deamidation Characterization of Monoclonal Antibody by LC/MS/MS. Anal. Chem. 2005, 77, 1432–1439.CrossRefGoogle Scholar
  27. 27.
    Di Donato, A. Ciardiello, M. A. Denigris, M. Piccoli, R. Mazzarella, L. Dalessio, G. Selective Deamidation of Ribonuclease-A Isolation and Characterization of the Resulting Isoaspartyl and Aspartyl Derivatives. J. Biol. Chem. 1993, 268, 4745–4751.Google Scholar
  28. 28.
    Solstad, T. Carvalho, R. N. Andersen, O. A. Waidelich, D. Flatmark, T. Deamidation of Labile Asparagine Residues in the Autoregulatory Sequence of Human Phenylalanine Hydroxylase—Structural and Functional Implications. Eur. J. Biochem. 2003, 270, 929–938.CrossRefGoogle Scholar
  29. 29.
    Smyth, D. G. Stein, W. H. Moore, S. On the Sequence of Residues 11 to 18 in Bovine Pancreatic Ribonuclease. J. Biol. Chem. 1962, 237, 1845–1850.Google Scholar
  30. 30.
    McLafferty, F. W. Tandem Mass Spectrometry; Wiley: New York, 1983.Google Scholar
  31. 31.
    Shukla, A. K. Futrell, J. H. Collisional Activation and Dissociation of Polyatomic Ions. Mass Spectrom. Rev. 1993, 12, 211–255.CrossRefGoogle Scholar
  32. 32.
    Lehmann, W. Schlosser, A. Erben, G. Pipkorn, R. Bossemeyer, D. Kinzel, V. Analysis of Isoaspartate in Peptides by Electrospray Tandem Mass Spectrometry. Protein Sci. 2000, 9, 2268–2290.Google Scholar
  33. 33.
    Gonzalez, L. J. Shimizu, T. Satomi, Y. Betancourt, L. Besada, V. Padron, G. Orlando, R. Shirasawa, T. Shimonishi, Y. Takao, T. Differentiating α- and β-Aspartic Acids by Electrospray Ionization and Low-Energy Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 2092–2102.CrossRefGoogle Scholar
  34. 34.
    Schindler, P. Muller, D. Marki, W. Grossenbacher, H. Richter, W. J. Characterization of a β-Asp33 Isoform of Recombinant Hirudin Sequence Variant 1 by Low-Energy Collision-Induced Dissociation. J. Mass Spectrom. 1996, 31, 967–974.CrossRefGoogle Scholar
  35. 35.
    Luu, N. C. Robinson, S. Zhao, R. McKean, R. Ridge, D. P. Mass Spectrometric Differentiation of α- and β-Aspartic Acid in a Pseudo-Tetrapeptide Thrombosis Inhibitor and Its Isomer. Eur. J. Mass Spectrom. 2004, 10, 279–287.CrossRefGoogle Scholar
  36. 36.
    Zubarev, R. A. Kelleher, N. L. McLafferty, F. W. Electron Capture Dissociation of Multiply Charged Protein Cations: A Nonergodic Process. J. Am. Chem. Soc. 1998, 120, 3265–3266.CrossRefGoogle Scholar
  37. 37.
    Cournoyer, J. J. Pittman, J. L. Ivleva, V. I. Fallows, E. Waskell, L. Costello, C. E. O’Connor, P. B. Deamidation: Differentiation of Aspartyl from Isoaspartyl Products in Peptides by Electron Capture Dissociation. Protein Sci. 2005, 14, 452–463.CrossRefGoogle Scholar
  38. 38.
    O’Connor, P. B. Cournoyer, J. J. Pitteri, S. J. Chrisman, P. A. McLuckey, S. A. Differentiation of Aspartic and Isoaspartic Acids Using Electron Transfer Dissociation. J. Am. Soc. Mass Spectrom. 2006, 17, 15–19.CrossRefGoogle Scholar
  39. 39.
    Stensballe, A. Jensen, O. N. Olsen, J. V. Haselmann, K. F. Zubarev, R. A. Electron Capture Dissociation of Singly and Multiply Phosphorylated Peptides. Rapid Commun. Mass Spectrom. 2000, 14, 1793–1800.CrossRefGoogle Scholar
  40. 40.
    Mirgorodskaya, E. Roepstorff, P. Zubarev, R. A. Localization of O-Glycosylation Sites in Peptides by Electron Capture Dissociation in a Fourier Transform Mass Spectrometer. Anal. Chem. 1999, 71, 4431–4436.CrossRefGoogle Scholar
  41. 41.
    Cooper, H. J. Hudgins, R. R. Hakansson, K. Marshall, A. G. Characterization of Amino Acid Side-Chain Losses in Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2002, 13, 241–249.CrossRefGoogle Scholar
  42. 42.
    Desaire, H. Leary, J. A. Detection and Quantification of the Sulfated Disaccharides in Chondroitin Sulfate by Electrospray Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 916–920.CrossRefGoogle Scholar
  43. 43.
    Hitchcock, A. M. Costello, C. E. Zaia, J. Glycoform Quantification of Chondroitin/Dermatan Sulfate Using a Liquid Chromatography-Tandem Mass Spectrometry Platform. Biochemistry 2006, 45, 2350–2361.CrossRefGoogle Scholar
  44. 44.
    O’Connor, P. B. Pittman, J. L. Thomson, B. A. Budnik, B. A. Cournoyer, J. J. Jebanathirajah, J. Lin, C. Moyer, S. Zhao, C. A New Hybrid Electrospray Fourier Transform Mass Spectrometer: Design and Performance Characteristics. Rapid Commun. Mass Spectrom. 2006, 20, 259–266.CrossRefGoogle Scholar
  45. 45.
    Jebanathirajah, J. A. Pittman, J. L. Thomson, B. A. Budnik, B. A. Kaur, P. Rape, M. Kirschner, M. Costello, C. E. O’Connor, P. B. Characterization of a New qQq-FTICR Mass Spectrometer for Post-Translational Modification Analysis and Top-Down Tandem Mass Spectrometry of Whole Proteins. J. Am. Soc. Mass Spectrom. 2005, 16, 1985–1999.CrossRefGoogle Scholar
  46. 46.
    Breuker, K. Oh, H. B. Horn, D. M. Cerda, B. A. McLafferty, F. W. Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation. J. Am. Chem. Soc. 2002, 124, 6407–6420.CrossRefGoogle Scholar
  47. 47.
    Lin, C.; Cournoyer, J. C.; O’Connor, P. B. Use of a Double Resonance Electron Capture Dissociation to Probe Fragment Intermediate Lifetimes. J. Am. Soc. Mass Spectrom. 2006.Google Scholar
  48. 48.
    Shimizu, T. Watanabe, A. Ogawara, M. Mori, H. Shirasawa, T. Isoaspartate Formation and Neurodegeneration in Alzheimer’s Disease. Arch. Biochem. Biophys. 2000, 381, 225–234.CrossRefGoogle Scholar
  49. 49.
    Zubarev, R. A. Horn, D. M. Fridriksson, E. K. Kelleher, N. L. Kruger, N. A. Lewis, M. A. Carpenter, B. K. McLafferty, F. W. Electron Capture Dissociation for Structural Characterization of Multiply Charged Protein Cations. Anal. Chem. 2000, 72, 563–573.CrossRefGoogle Scholar
  50. 50.
    Iavarone, A. T. Paech, K. Williams, E. R. Effects of Charge State and Cationizing Agent on the Electron Capture Dissociation of a Peptide. Anal. Chem. 2004, 76, 2231–2238.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Jason J. Cournoyer
    • 1
    • 2
  • Cheng Lin
    • 2
  • Michael J. Bowman
    • 2
  • Peter B. O’Connor
    • 2
  1. 1.Department of ChemistryBoston UniversityBostonUSA
  2. 2.Department of Biochemistry, Mass Spectrometry ResourceBoston University School of MedicineBostonUSA

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