Recognition and quantification of binary and ternary mixtures of isomeric peptides by the kinetic method: metal ion and ligand effects on the dissociation of metal-bound complexes

  • Lianming Wu
  • Karel Lemr
  • Tenna Aggerholm
  • R. Graham Cooks


The kinetic method is applied to differentiate and quantify mixtures of isomeric tripeptides based on the competitive dissociations of divalent metal ion-bound clusters in an ion trap mass spectrometer. This methodology is extended further to determine compositions of ternary mixtures of the isomers Gly-Gly-Ala (GGA), Ala-Gly-Gly (AGG), and Gly-Ala-Gly (GAG). This procedure also allows to perform chiral quantification of a ternary mixture of optical isomers. The divalent metal ion CaII is particularly appropriate for isomeric distinction and quantification of the isobaric tripeptides Gly-Gly-Leu/Gly-Gly-Ile (GGL/GGI). Among the first-row transition metal ions, CuII yields remarkably effective isomeric differentiation for both the isobaric tripeptides, GGI/GGL using GAG as the reference ligand, and the positional isomers GAG/GGA using GGI as the reference ligand. This is probably due to agostic bonding: α-agostic bonding occurs between CuII and GAG and β-agostic bonding between CuII and GGI, each produces large but different steric effects on the stability of the CuII-bound dimeric clusters. These data form the basis for possible future quantitative analyses of mixtures of larger peptides such as are generated, for example, in combinatorial synthesis of peptides and peptide mimics.


  1. 1.
    Sigel, H.; Sigel, A. Metal Ion in Biological Systems Vol. XXXIII. Vanadium and Its Role in Life Dekker: New York, 1995; pp 759.Google Scholar
  2. 2.
    Brittain, I. J.; Huang, X.; Long, E. C. Selective Recognition and Cleavage of RNA Loop Structures by Ni(II)-Xaa-Gly-His Metallopeptides. Biochem 1998, 37, 12113–12120.CrossRefGoogle Scholar
  3. 3.
    Atwood, C. S.; Moir, R. D.; Huang, X.; Scarpa, R. C.; Bacarra, N. M. E.; Romano, D. M.; Hartshorn, M. A.; Tanzi, R. E.; Bush, A. I. Dramatic Aggregation of Alzheimer Aβ by Cu(II) is Induced by Conditions Representing Physiological Acidosis. J. Biol. Chem. 1998, 273, 12817–12826.CrossRefGoogle Scholar
  4. 4.
    Moore, G. J. Discovery and Design of Peptide Mimetics. Proc. West. Pharmacol. Soc. 1997, 40, 115–119.Google Scholar
  5. 5.
    Olivera, B. M.; Hillyard, D. R.; Marsh, M.; Yoshikami, D. Combinatorial Peptide Libraries in Drug Design: Lessons from Venomous Cone Snails. Trends Biotechnol 1995, 13, 422–426.CrossRefGoogle Scholar
  6. 6.
    Barnes, C. A. S.; Hilderbrand, A. E.; Valentine, S. J.; Clemmer, D. E. Resolving Isomeric Peptide Mixtures: A Combined HPLC/Ion Mobility-TOFMS Analysis of a 4000 Component Combinatorial Library. Anal. Chem. 2001, 74, 26–36.CrossRefGoogle Scholar
  7. 7.
    Larsen, M. R.; Roepstorff, P. Mass Spectrometric Identification of Proteins and Characterization of Their Post-Translational Modifications in Proteome Analysis. Fresen. Anal. Chem. 2000, 366, 677.CrossRefGoogle Scholar
  8. 8.
    Fenn, J. B.; Mann, N.; Meng, C. K.; Wong, S. F. Electrospray Ionization—;Principles and Practice. Mass Spectrom. Rev. 1990, 9, 37–70.CrossRefGoogle Scholar
  9. 9.
    Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Biopolymers. Anal. Chem. 1991, 63, 1193A-1203A.CrossRefGoogle Scholar
  10. 10.
    Roepstorff, P.; Richter, W. J. Status of, and Developments in, Mass Spectrometry of Peptides and Proteins. J. Mass Spectrom. Ion Processes 1992, 118/119, 789–809.CrossRefGoogle Scholar
  11. 11.
    Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry. VCH Publishers: New York, 1988, pp 1–12, 173–178.Google Scholar
  12. 12.
    Wee, S.; O’Hair, R. A. J.; McFadyen, W. D. Side-Chain Radical Losses from Radical Cations Allows Distinction of Leucine and Isoleucine Residues in the Isomeric Peptides Gly-XXX-Arg. Rapid Commun. Mass Spectrom. 2002, 16, 884–890.CrossRefGoogle Scholar
  13. 13.
    Nemirovskiy, O. V.; Gross, M. L. Determination of Calcium Binding Sites in Gas-Phase Small Peptides by Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1998, 9, 1020–1028.CrossRefGoogle Scholar
  14. 14.
    Nemirovskiy, O. V.; Gross, M. L. Intrinsic Ca2+ Affinities of Peptides: Application of the Kinetic Method to Analogs of Calcium-Binding Site III of Rabbit Skeletal Troponin C. J. Am. Soc. Mass Spectrom. 2000, 11, 770–779.CrossRefGoogle Scholar
  15. 15.
    Cooks, R. G.; Ast, T.; Pradeep, T.; Wysocki, V. Reactions of Ions with Organic Surfaces. Acc. Chem. Res. 1994, 27, 316–323.CrossRefGoogle Scholar
  16. 16.
    Cox, A. L.; Skipper, J.; Chen, Y.; Henderson, R. A.; Darrow, T. L.; Sharbanowitz, J.; Engelhard, V. H.; Hunt, D. F.; Slingluff, C. L. Identification of a Peptide Recognized by Five Melanoma-Specific Human Cytotoxic T Cell Lines. Science 1994, 264, 716–719.CrossRefGoogle Scholar
  17. 17.
    Lippincott, J.; Fattor, T. J.; Lemon, D. D.; Apostol, I. Application of Native-State Electrospray Mass Spectrometry to Identify Zinc-Binding Sites on Engineered Hemoglobin. Anal. Biochem. 2000, 284, 247–255.CrossRefGoogle Scholar
  18. 18.
    Mohammed, S.; Chalmers, M. J.; Gielbert, J.; Ferro, M.; Gora, L.; Smith, D. C.; Gaskell, S. J. A Novel Tandem Quadrupole Mass Spectrometer Allowing Gaseous Collisional Activation and Surface Induced Dissociation. J. Mass Spectrom. 2001, 36, 1260–1268.CrossRefGoogle Scholar
  19. 19.
    Jonsson, A. P.; Bergman, T.; Jornvall, H.; Griffiths, W. J.; Bratt, P.; Stromberg, N. Gln-Gly Cleavage: Correlation between Collision-Induced Dissociation and Biological Degradation. J. Am. Soc. Mass Spectrom. 2001, 12, 337–342.CrossRefGoogle Scholar
  20. 20.
    Cooks, R. G.; Kruger, T. L. Intrinsic Basicity Determination Using Metastable Ions. J. Am. Chem. Soc. 1977, 99, 1279–1281.CrossRefGoogle Scholar
  21. 21.
    Cooks, R. G.; Patrick, J. S.; Kotiaho, T.; McLuckey, S. A. Thermochemical Determinations by the Kinetic Method. Mass Spectrom. Rev. 1994, 13, 287–339.CrossRefGoogle Scholar
  22. 22.
    Cooks, R. G.; Wong, P. S. H. Kinetic Method of Making Thermochemical Determinations. Acc. Chem. Res. 1998, 31, 379–386.CrossRefGoogle Scholar
  23. 23.
    Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. Copper(II)-Assisted Enantiomeric Analysis of D,L-Amino Acids Using the Kinetic Method: Chiral Recognition and Quantification in the Gas Phase. J. Am. Chem. Soc. 2000, 122, 10598–10609.CrossRefGoogle Scholar
  24. 24.
    Tao, W. A.; Wu, L.; Cooks, R. G. Rapid Enantiomeric Quantitation of an Antiviral Nucleoside Agent (D,L-FMAU, 2′-Fluoro-5-Methyl-beta, D,L-Arabinofuranosyluracil) by Mass Spectrometry. J. Med. Chem. 2001, 44, 3541–3544.CrossRefGoogle Scholar
  25. 25.
    Wu, L.; Tao, W. A.; Cooks, R. G. Ligand and Metal Ion Effects in Metal Ion Clusters Used for Chiral Analysis of α-Hydroxy Acids by the Kinetic Method. Anal. Bioanal. Chem. 2002, 373, 618–627.CrossRefGoogle Scholar
  26. 26.
    Tao, W. A.; Wu, L.; Cooks, R. G. Differentiation and Quantitation of Isomeric Dipeptides by Low-Energy Dissociation of Copper(II)-Bound Complexes. J. Am. Soc. Mass Spectrom. 2001, 12, 490–496.CrossRefGoogle Scholar
  27. 27.
    Ryzhov, V.; Dunbar, R. C.; Cerda, B.; Wesdemiotis, C. Cation—π Effects in the Complexation of Na+ and K+ with Phe, Tyr, and Trp in the Gas Phase. J. Am. Soc. Mass Spectrom. 2000, 11, 1037–1046.CrossRefGoogle Scholar
  28. 28.
    Cerda, B. A.; Wesdemiotis, C. Zwitterionic versus Charge-Solvated Structures in the Binding of Arginine to Alkali Metal Ions in the Gas Phase. Analyst 2000, 125, 657–660.CrossRefGoogle Scholar
  29. 29.
    Wang, F.; Ma, S.; Wong, P.; Cooks, R. G.; Gozzo, F. C.; Eberlin, M. N. Gas Phase Agostic Bonding in Pyridine SiFn+ (n = 1, 3) Cluster Ions Investigated by the Kinetic Method. Int. J. Mass Spectrom. 1998, 179/180, 195–205.CrossRefGoogle Scholar
  30. 30.
    Timmers, F.; Brookhart, M. Synthesis and Reactivity of Methyl-Substituted Butenylmanganese Tricarbonyl Complexes Containing a Two-Electron, Three-Center Mn·H·C Interaction. Organometallics 1985, 4, 1365–1371.CrossRefGoogle Scholar
  31. 31.
    Cooks, R. G.; Rockwood, A. L. The “Thomson”. A suggested Unit for Mass Spectroscopists. Rapid Commun. Mass Spectrom. 1991, 5, 93.Google Scholar
  32. 32.
    Laskin, J.; Futrell, J. H. The Theoretical Basis of the Kinetic Method from the Point of View of Finite Heat Bath Theory. J. Phys. Chem. A 2000, 104, 8829–8837.CrossRefGoogle Scholar
  33. 33.
    Tao, W. A.; Zhang, D.; Wang, F.; Thomas, P.; Cooks, R. G. Kinetic Resolution of D, L-Amino Acids Based on Gas-Phase Dissociation of Copper(II) Complexes. Anal. Chem. 1999, 71, 4427–4429.CrossRefGoogle Scholar
  34. 34.
    Zhang, D.; Tao, W. A.; Cooks, R. G. Chiral Resolution of D- and L-Amino Acids by Tandem Mass Spectrometry of Ni(II)-Bound Trimeric Complexes. Int. J. Mass Spectrom. 2001, 204, 159–169.CrossRefGoogle Scholar
  35. 35.
    Tao, W. A.; Wu, L.; Cooks, R. G. Rapid Enantiomeric Determination of α-Hydroxy Acids by Electrospray Ionization Tandem Mass Spectrometry. Chem. Commun. 2000, 20, 2023–2024.CrossRefGoogle Scholar
  36. 36.
    Tao, W. A.; Cooks, R. G. Parallel Reactions for Enantiomeric Quantification of Peptides by Mass Spectrometry. Angew. Chem. Int. Ed. 2001, 40, 757–760.CrossRefGoogle Scholar
  37. 37.
    Augusti, D. V.; Carazza, F.; Augusti, R.; Tao, W. A.; Cooks, R. G. Quantitative Chiral Analysis of Sugars by Electrospray Ionization Tandem Mass Spectrometry Using Modified Amino Acids as Chiral Reference Compounds. Anal. Chem. 2002, 74, 3458–3462.CrossRefGoogle Scholar
  38. 38.
    Wu, L., Cooks, R. G. Chiral Analysis Using the Kinetic Method with Optimized Fixed Ligands: Application to Oxazolidinone Antibiotics. Anal. Chem. in pressGoogle Scholar
  39. 39.
    Wu, L. Tao, W. A., Cooks, R. G. Kinetic Method for Simultaneous Chiral Analysis of Different Amino Acids in Mixtures. J. Mass Spectrom. in pressGoogle Scholar
  40. 40.
    Harvey, D. J. Ionization and Collision-Induced Fragmentation of N-linked and Related Carbohydrates Using Divalent Cations. J. Am. Soc. Mass Spectrom. 2001, 12, 926–937.CrossRefGoogle Scholar
  41. 41.
    Wu, L., Clark, R. L., Cooks, R. G. Chiral Quantification of D-, L-, and meso-Tartaric Acid Mixtures Using a Mass Spectrometric Kinetic Method. Chem. Commun. 2003, 137, 136–137.CrossRefGoogle Scholar
  42. 42.
    Carr, N.; Mole, L.; Orpen, A. G.; Spencer, J. L. Platinum Ethyl Complexes with β-Agostic Platinum-Hydrogen-Carbon Bonding. J. Chem. Soc. Dalton Trans. 1992, 18, 2653–2662.CrossRefGoogle Scholar
  43. 43.
    Jahn, H. A.; Teller, E. Stability of Polyatomic Molecules in Degenerate Electronic States. I. Orbital Degeneracy. Proc. Roy. Soc. London A 1937, A161, 220–235.Google Scholar
  44. 44.
    Irving, H.; Williams, R. J. P. Order of Stability of Metal Complexes. Nature 1948, 162, 746–747.CrossRefGoogle Scholar
  45. 45.
    Sigel, H.; Martin, R. B. Coordinating Properties of the Amide Bond. Stability and Structure of Metal Ion Complexes of Peptides and Related Ligands. Chem. Rev. 1982, 82, 385–426.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2003

Authors and Affiliations

  • Lianming Wu
    • 1
  • Karel Lemr
    • 1
  • Tenna Aggerholm
    • 1
  • R. Graham Cooks
    • 1
  1. 1.Department of ChemistryPurdue UniversityWest LafayetteUSA

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