Use of 18O labels to monitor deamidation during protein and peptide sample processing

  • Xiaojuan Li
  • Jason J. Cournoyer
  • Cheng Lin
  • Peter B. O’Connor
Articles

Abstract

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c · + 58 and z − 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)(n−1)+· − 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of 18O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (∼4 h) trypsin digestion process generates no additional detectable peptide deamidations.

Supplementary material

13361_2011_190600855_MOESM1_ESM.doc (298 kb)
Supplementary material, approximately 306 KB.

References

  1. 1.
    Robinson, N. E.; Robinson, A. B. Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins. Althouse Press: Cave Junction, OR, 2004.Google Scholar
  2. 2.
    Robinson, N. E. Protein Deamidation. Proc. Natl. Acad. Sci. U. S. A.. 2002, 99, 5283–5288.CrossRefGoogle Scholar
  3. 3.
    Robinson, N. E.; Robinson, A. B. Molecular Clocks. Proc. Natl. Acad. Sci. U. S. A.. 2001, 98, 944–949.CrossRefGoogle Scholar
  4. 4.
    Lindner, H.; Sarg, B.; Hoertnagl, B.; Helliger, W. The Microheterogeneity of the Mammalian H1(0) Histone—Evidence for an Age-Dependent Deamidation. J. Biol. Chem. 1998, 273, 13324–13330.CrossRefGoogle Scholar
  5. 5.
    Bischoff, R.; Kolbe, H. V. Deamidation of Asparagine and Glutamine Residues in Proteins and Peptides—Structural Determinants and Analytical Methodology. J. Chromatogr. B Biomed. Appl. 1994, 662, 261–278.CrossRefGoogle Scholar
  6. 6.
    Lindner, H.; Helliger, W. Age-Dependent Deamidation of Asparagine Residues in Proteins. Exp. Gerontol. 2001, 36, 1551–1563.CrossRefGoogle Scholar
  7. 7.
    Capasso, S. Estimation of the Deamidation Rate of Asparagine Side Chains. J. Pept. Res. 2000, 55, 224–229.CrossRefGoogle Scholar
  8. 8.
    Geiger, T.; Clarke, S. Deamidation, Isomerization, and Racemization at Asparaginyl and Aspartyl Residues in Peptides—Succinimide-Linked Reactions That Contribute to Protein-Degradation. J. Biol. Chem. 1987, 262, 785–794.Google Scholar
  9. 9.
    Xie, M. L.; Schowen, R. L. Secondary Structure and Protein Deamidation. J. Pharm. Sci. 1999, 88, 8–13.CrossRefGoogle Scholar
  10. 10.
    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
  11. 11.
    Tylercross, 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
  12. 12.
    Wright, H. T. Nonenzymatic Deamidation of Asparaginyl and Glutaminyl Residues in Proteins. Crit. Rev. Biochem. Mol. Biol. 1991, 26, 1–52.CrossRefGoogle Scholar
  13. 13.
    Kossiakoff, A. A. Tertiary Structure Is a Principal Determinant to Protein Deamidation. Science. 1988, 240, 191–194.CrossRefGoogle Scholar
  14. 14.
    Peters, B.; Trout, B. L. Asparagine Deamidation: pH-Dependent Mechanism from Density Functional Theory. Biochemistry. 2006, 45, 5384–5392.CrossRefGoogle Scholar
  15. 15.
    Joshi, A. B.; Sawai, M.; Kearney, W. R.; Kirsch, L. E. Studies on the Mechanism of Aspartic Acid Cleavage and Glutamine Deamidation in the Acidic Degradation of Glucagon. J. Pharm. Sci. 2005, 94, 1912–1927.CrossRefGoogle Scholar
  16. 16.
    Robinson, N. E.; Zabrouskov, V.; Zhang, J.; Lampi, K. J.; Robinson, A. B. Measurement of Deamidation of Intact Proteins by Isotopic Envelope and Mass Defect with Ion Cyclotron Resonance Fourier Transform Mass Spectrometry. Rapid Commun. Mass Spectrom. 2006, 20, 3535–3541.CrossRefGoogle Scholar
  17. 17.
    Robinson, N. E.; Lampi, K. J.; Speir, J. P.; Kruppa, G.; Easterling, M.; Robinson, A. B. Quantitative Measurement of Young Human Eye Lens Crystallins by Direct Injection Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Mol. Vis. 2006, 12, 704–711.Google Scholar
  18. 18.
    Robinson, N. E.; Lampi, K. J.; McIver, R. T.; Williams, R. H.; Muster, W. C.; Kruppa, G.; Robinson, A. B. Quantitative Measurement of Deamidation in Lens Beta B2-Crystallin and Peptides by Direct Electrospray Injection and Fragmentation in a Fourier Transform Mass Spectrometer. Mol. Vis. 2005, 11, 1211–1219.Google Scholar
  19. 19.
    Schmid, D. G.; von der Mulbe, F.; Fleckenstein, B.; Weinschenk, T.; Jung, G. Broadband Detection Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry to Reveal Enzymatically and Chemically Induced Deamidation Reactions within Peptides. Anal. Chem. 2001, 73, 6008–6013.CrossRefGoogle Scholar
  20. 20.
    Chazin, W. J.; Kordel, J.; Thulin, E.; Hofmann, T.; Drakenberg, T.; Forsen, S. Identification of an Isoaspartyl Linkage Formed upon Deamidation of Bovine Calbindin-D9k and Structural Characterization by 2D 1H NMR. Biochemistry. 1989, 28, 8646–8653.CrossRefGoogle Scholar
  21. 21.
    Carlson, 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
  22. 22.
    Carr, S. A.; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Integration of Mass-Spectrometry in Analytical Biotechnology. Anal. Chem. 1991, 63, 2802–2824.CrossRefGoogle Scholar
  23. 23.
    Zhang, W.; Czupryn, M. J. Analysis of Isoaspartate in a Recombinant Monoclonal Antibody and Its Charge Isoforms. J. Pharm. Biomed. Anal. 2003, 30, 1479–1490.CrossRefGoogle Scholar
  24. 24.
    Gonzalez, L. J.; Shimizu, T.; Satomi, Y.; Betancourt, L.; Besada, V.; Padron, G.; Orlando, R.; Shirasawa, T.; Shimonishi, Y.; Takao, T. Differentiating Alpha- and Beta-Aspartic Acids by Electrospray Ionization and Low-Energy Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 2092–2102.CrossRefGoogle Scholar
  25. 25.
    Castet, S.; Enjalbal, C.; Fulcrand, P.; Guichou, J. F.; Martinez, J.; Aubagnac, J. L. Characterization of Aspartic Acid and Beta-Aspartic Acid in Peptides by Fast-Atom Bombardment Mass Spectrometry and Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 1934–1938.CrossRefGoogle Scholar
  26. 26.
    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
  27. 27.
    Zubarev, R. A.; Haselmann, K. F.; Budnik, B.; Kjeldsen, F.; Jensen, F. Towards an Understanding of the Mechanism of Electron-Capture Dissociation: A Historical Perspective and Modern Ideas. Eur. J. Mass Spectrom. 2002, 8, 337–349.CrossRefGoogle Scholar
  28. 28.
    Syrstad, E. A.; Turecek, F. Toward a General Mechanism of Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2005, 16, 208–224.CrossRefGoogle Scholar
  29. 29.
    Leymarie, N.; Costello, C. E.; O’Connor, P. B. Electron Capture Dissociation Initiates a Free Radical Reaction Cascade. J. Am. Chem. Soc. 2003, 125, 8949–8958.CrossRefGoogle Scholar
  30. 30.
    Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F. Anion Dependence in the Partitioning between Proton and Electron Transfer in Ion/Ion Reactions. Int. J. Mass Spectrom. 2004, 236, 33–42.CrossRefGoogle Scholar
  31. 31.
    Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528–9533.CrossRefGoogle Scholar
  32. 32.
    Cournoyer, J. J.; Lin, C.; Bowman, M. J.; O’Connor, P. B. Quantitating the Relative Abundance of Isoaspartyl Residues in Deamidated Proteins by Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2007, 18, 48–56.CrossRefGoogle Scholar
  33. 33.
    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
  34. 34.
    Cournoyer, J. J.; Pittman, J. L.; Ivleva, V. B.; 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
  35. 35.
    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
  36. 36.
    Horn, D. M.; Ge, Y.; McLafferty, F. W. Activated Ion Electron Capture Dissociation for Mass Spectral Sequencing of Larger (42 kDa) Proteins. Anal. Chem. 2000, 72, 4778–4784.CrossRefGoogle Scholar
  37. 37.
    Yao, X. D.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Proteolytic O-18 Labeling for Comparative Proteomics: Model Studies with Two Serotypes of Adenovirus. Anal. Chem. 2004, 76, 2675–2675.CrossRefGoogle Scholar
  38. 38.
    Stewart, I. I.; Thomson, T.; Figeys, D. O-18 Labeling: A Tool for Proteomics. Rapid Commun. Mass Spectrom. 2001, 15, 2456–2465.CrossRefGoogle Scholar
  39. 39.
    Heller, M.; Mattou, H.; Menzel, C.; Yao, X. D. Trypsin Catalyzed O-16-to-O-18 Exchange for Comparative Proteomics: Tandem Mass Spectrometry Comparison Using MALDI-TOF, ESI-QTOF, and ESI-Ion Trap Mass Spectrometers. J. Am. Soc. Mass Spectrom. 2003, 14, 704–718.CrossRefGoogle Scholar
  40. 40.
    Yao, X. D.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Proteolytic O-18 Labeling for Comparative Proteomics: Model Studies with Two Serotypes of Adenovirus. Anal. Chem. 2001, 73, 2836–2842.CrossRefGoogle Scholar
  41. 41.
    Miyagi, M.; Rao, K. C. S. Proteolytic O-18-Labeling Strategies for Quantitative Proteomics. Mass Spectrom. Rev. 2007, 26, 121–136.CrossRefGoogle Scholar
  42. 42.
    Katta, V.; Chait, B. T. Hydrogen/Deuterium Exchange Electrospray Ionization Mass Spectrometry: A Method for Probing Protein Conformational Changes in Solution. J. Am. Chem. Soc. 1993, 115, 6317–6321.CrossRefGoogle Scholar
  43. 43.
    Wales, T. E.; Engen, J. R. Hydrogen Exchange Mass Spectrometry for the Analysis of Protein Dynamics. Mass Spectrom. Rev. 2006, 25, 158–170.CrossRefGoogle Scholar
  44. 44.
    Garcia, R. A.; Pantazatos, D.; Villarreal, F. J. Hydrogen/Deuterium Exchange Mass Spectrometry for Investigating Protein-Ligand Interactions. Assay Drug. Dev. Technol. 2004, 2, 81–91.CrossRefGoogle Scholar
  45. 45.
    Kheterpal, I.; Cook, K. D.; Wetzel, R. Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of Protein Aggregates. Methods Enzymol. 2006, 413, 140–166.CrossRefGoogle Scholar
  46. 46.
    Kheterpal, I.; Wetzel, R. Hydrogen/Deuterium Exchange Mass Spectrometries—A Window into Amyloid Structure. Acc. Chem. Res. 2006, 39, 584–593.CrossRefGoogle Scholar
  47. 47.
    Xiao, G.; Bondarenko, P. V.; Jacob, J.; Chu, G. C.; Chelius, D. O18 Labeling Method for Identification and Quantification of Succinimide in Proteins. Anal. Chem. 2007, 79, 2714–2721.CrossRefGoogle Scholar
  48. 48.
    Kubota, K.; Yoneyama-Takazawa, T.; Ichikawa, K. Determination of Sites Citrullinated by Peptidylarginine Deiminase Using O-18 Stable Isotope Labeling and Mass Spectrometry. Rapid Commun. Mass Spectrom. 2005, 19, 683–688.CrossRefGoogle Scholar
  49. 49.
    Schnolzer, M.; Jedrzejewski, P.; Lehmann, W. D. Protease-Catalyzed Incorporation of O-18 into Peptide Fragments and Its Application for Protein Sequencing by Electrospray and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Electrophoresis. 1996, 17, 945–953.CrossRefGoogle Scholar
  50. 50.
    Antonov, V. K.; Ginodman, L. M.; Rumsh, L. D.; Kapitannikov, Y. V.; Barshevskaya, T. N.; Yavashev, L. P.; Gurova, A. G.; Volkova, L. I. Studies on the Mechanisms of Action of Proteolytic Enzymes Using Heavy Oxygen Exchange. Eur. J. Biochem. 1981, 117, 195–200.CrossRefGoogle Scholar
  51. 51.
    O’Connor, P. B.; Pittman, J. L.; Thomson, B. A.; Budnik, B. A.; Cournoyer, J. C.; 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
  52. 52.
    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
  53. 53.
    Tsybin, Y. O.; Witt, M.; Baykut, G.; Hakansson, P. Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in the Electron Energy Range 0–50 eV. Rapid Commun. Mass Spectrom. 2004, 18, 1607–1613.CrossRefGoogle Scholar
  54. 54.
    Tsybin, Y. O.; Hakansson, P.; Budnik, B. A.; Haselmann, K. F.; Kjeldsen, F.; Gorshkov, M.; Zubarev, R. A. Improved Low-Energy Electron Injection Systems for High Rate Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Rapid Commun. Mass Spectrom. 2001, 15, 1849–1854.CrossRefGoogle Scholar
  55. 55.
    Tsybin, Y. O.; Ramstrom, M.; Witt, M.; Baykut, G.; Hakansson, P. Peptide and Protein Characterization by High-Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Mass Spectrom. 2004, 39, 719–729.CrossRefGoogle Scholar
  56. 56.
    Cheung, W. Y. Calmodulin Plays a Pivotal Role in Cellular Regulation. Science. 1980, 207, 19–27.CrossRefGoogle Scholar
  57. 57.
    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 Domains II, III, and IV. Protein Sci. 1993, 2, 1648–1663.CrossRefGoogle Scholar
  58. 58.
    Zubarev, R. A. Electron-Capture Dissociation Tandem Mass Spectrometry. Curr. Opin. Biotechnol. 2004, 15, 12–16.CrossRefGoogle Scholar
  59. 59.
    Yergey, J. A. A general approach to calculating isotopic distributions for mass spectrometry. Int. J. Mass Spectrom. Ion Processes. 1983, 52, 337–349.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Xiaojuan Li
    • 1
  • Jason J. Cournoyer
    • 2
  • Cheng Lin
    • 1
  • Peter B. O’Connor
    • 1
  1. 1.Mass Spectrometry Resource, Department of BiochemistryBoston University School of MedicineBostonUSA
  2. 2.Department of ChemistryBoston UniversityBostonUSA

Personalised recommendations