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Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

  • Oscar Yanes
  • Francesc X. Aviles
  • Peter Roepstorff
  • Thomas J. D. Jørgensen
Article

Abstract

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, “intensity fading MS” has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-µM range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower high-mass sensitivity of MCP detectors.

Keywords

MALDI Noncovalent Interaction Sinapic Acid Noncovalent Complex MALDI Mass Spectrum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Matrix-Assisted Laser Desorption Ionization Mass-Spectrometry of Biopolymers. Anal. Chem. 1991, 63, A1193-A1202.CrossRefGoogle Scholar
  2. 2.
    Karas, M.; Bahr, U. Laser Desorption Ionization Mass-Spectrometry of Large Biomolecules. Trac-Trends in Anal. Chem. 1990, 9, 321–325.CrossRefGoogle Scholar
  3. 3.
    Jespersen, S.; Niessen, W. M. A.; Tjaden, U. R.; Van der Greef, J. Basic Matrices in the Analysis of Non-Covalent Complexes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Mass Spectrom. 1998, 33, 1088–1093.CrossRefGoogle Scholar
  4. 4.
    Moniatte, M.; Lesieur, C.; Vécsey-Semjén, B.; Buckley, J. T.; Pattus, F.; Van der Goot, F. G.; Van Dorsselaer, A. Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry in the Subunit Stoichmetry Study of High-Mass Non-Covalent Complexes. Int. J. Mass Spectrom. Ion Processes 1997, 169, 179–199.CrossRefGoogle Scholar
  5. 5.
    Tang, X.; Callahan, J. H.; Zhou, P.; Vertes, A. Noncovalent Protein-Oligonucleotide Interactions Monitored by Matrix-Assisted Laser Desorption/Ionization Mass-Spectrometry. Anal Chem. 1995, 67, 4542–4548.CrossRefGoogle Scholar
  6. 6.
    Schlosser, G.; Pocsfalvi, G.; Malorni, A.; Puerta, A.; de Frutos, M.; Vekey, K. Detection of Immune Complexes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 2741–2747.CrossRefGoogle Scholar
  7. 7.
    Zehl, M.; Allmaier, G. Investigation of Sample Preparation and Instrumental Parameters in the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Noncovalent Peptide/Peptide Complexes. Rapid Commun. Mass Spectrom. 2003, 17, 1931–1940.CrossRefGoogle Scholar
  8. 8.
    Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbusch, J.; Dencher, N.; Krüger, U.; Galla, H. J. Matrix-Assisted Laser Desorption/Ionization Mass-Spectrometry (Maldi-MS) of Membrane-Proteins and Noncovalent Complexes. J. Mass Spectrom. 1995, 30, 1462–1468.CrossRefGoogle Scholar
  9. 9.
    Zehl, M.; Allmaier, G. Ultraviolet Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry of Intact Hemoglobin Complex from Whole Human Blood. Rapid Commun. Mass Spectrom. 2004, 18, 1932–1938.CrossRefGoogle Scholar
  10. 10.
    Luo, S. Z.; Li, Y. M.; Qiang, W.; Zhao, Y. F.; Abe, H.; Nemoto, T.; Qin, X. R.; Nakanishi, H. Detection of Specific Noncovalent Interaction of Peptide with DNA by Maldi-TOF. J. Am. Soc. Mass Spectrom. 2004, 15, 28–31.CrossRefGoogle Scholar
  11. 11.
    Woods, A. S.; Huestis, M. A. A Study of Peptide-Peptide Interaction by Matrix-Assisted Laser Desorption/Ionization. J. Am. Soc. Mass Spectrom. 2001, 12, 88–96.CrossRefGoogle Scholar
  12. 12.
    Woods, A. S.; Buchsbaum, J. C.; Worrall, T. A.; Berg, J. M.; Cotter, R. J. Matrix-Assisted Laser Desorption/Ionization of Noncolavently Bound Compounds. Anal. Chem. 1995, 67, 4462–4465.CrossRefGoogle Scholar
  13. 13.
    Cohen, L. R. H.; Strupat, K.; Hillenkamp, F. Analysis of Quaternary Protein Ensembles by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 1046–1052.CrossRefGoogle Scholar
  14. 14.
    Glocker, M. O.; Bauer, S. H.; Kast, J.; Volz, J.; Przybylski, M. Characterization of Specific Noncovalent Protein Complexes by UV Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. J. Mass Spectrom. 1996, 31, 1221–1227.CrossRefGoogle Scholar
  15. 15.
    Farmer, T. B.; Caprioli, R. M. Determination of Protein-Protein Interactions by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Mass Spectrom. 1998, 33, 697–704.CrossRefGoogle Scholar
  16. 16.
    Moniatte, M.; Van der Goot, F. G.; Buckley, J. T.; Pattus, F.; Van Dorsselaer, A. Characterization of the Heptameric Pore-Forming Complex of the Aeromonas Toxin Aerolysin using Maldi-TOF Mass Spectrometry. FEBS Lett. 1996, 384, 269–272.CrossRefGoogle Scholar
  17. 17.
    Zehl, M.; Allmaier, G. Instrumental Parameters in the Maldi-TOF Mass Spectrometric Analysis of Quaternary Protein Structures. Anal. Chem. 2005, 77, 103–110.CrossRefGoogle Scholar
  18. 18.
    Perera, I. K.; Allwood, D.; Dyer, P. E.; Oldershaw, G. A. Formation of Homo and Hetero Multimeric Ions of Large Proteins in Matrix-Assisted UV Laser Desorption Ionization. J. Mass Spectrom. 1995, Suppl. S3-S12.Google Scholar
  19. 19.
    Gruic-Sovulj, I.; Ludemann, H. C.; Hillenkamp, F.; Kucan, I.; Peter-Katalinic, J. Detection of Noncovalent tRNA-Aminoacyl-tRNA Synthetase Complexes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Biol. Chem. 1997, 272, 32084–32091.CrossRefGoogle Scholar
  20. 20.
    Vogl, T.; Roth, J.; Sorg, C.; Hillenkamp, F.; Strupat, K. Calcium-Induced Noncovalently Linked Tetramers of Mrp8 And Mrp14 Detected by Ultraviolet Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1999, 10, 1124–1130.CrossRefGoogle Scholar
  21. 21.
    Benesch, J. L.; Robinson, C. V. Mass Spectrometry of Macromolecular Assemblies: Preservation and Dissociation. Curr. Opin. Struct. Biol. 2006, 16, 245–251.CrossRefGoogle Scholar
  22. 22.
    Heck, A. J. R.; Van den Heuvel, R. H. H. Investigation of Intact Protein Complexes by Mass Spectrometry. Mass Spectrom. Rev. 2004, 23, 368–389.CrossRefGoogle Scholar
  23. 23.
    Livadaris, V.; Blais, J. C.; Tabet, J. C. Formation of Non-Specific Protein Cluster Ions in Matrix-Assisted Laser Desorption/Ionization: Abundances and Dynamical Aspects. Eur. J. Mass Spectrom. 2000, 6, 409–413.CrossRefGoogle Scholar
  24. 24.
    Perera, I. K.; Allwood, D.; Dyer, P. E.; Oldershaw, G. A. Observation of Mixed Molecular Cluster Ions in Matrix-Assisted UV Laser-Desorption Ionization of High-Mass Protein Mixtures. Int. J. Mass Spectrom. Ion Processes 1995, 145, L9.Google Scholar
  25. 25.
    Villanueva, J.; Yanes, O.; Querol, E.; Serrano, L.; Avilés, F. X. Identification of Protein Ligands in Complex Biological Samples using Intensity-Fading Maldi-TOF Mass Spectrometry. Anal. Chem. 2003, 75, 3385–3395.CrossRefGoogle Scholar
  26. 26.
    Kiselar, J. G.; Downard, K. M. Direct Identification of Protein Epitopes by Mass Spectrometry without Immobilization of Antibody and Isolation of Antibody-Peptide Complexes. Anal. Chem. 1999, 71, 1792–1801.CrossRefGoogle Scholar
  27. 27.
    Kiselar, J. G.; Downard, K. M. Antigenic Surveillance of the Influenza Virus by Mass Spectrometry. Biochemistry 1999, 38, 14185–14191.CrossRefGoogle Scholar
  28. 28.
    Yanes, O.; Villanueva, J.; Querol, E.; Avilés, F. X. Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading Maldi-TOF MS. Mol. Cell. Proteom. 2005, 4, 1602–1613.CrossRefGoogle Scholar
  29. 29.
    Yanes, O.; Villanueva, J.; Querol, E.; Avilés, F. X. Intensity-fading Maldi-Tof MS: Novel Screening for Ligand Binding and Drug Discovery. Drug Discov. Today: TARGETS 2004, 3, 23–30.CrossRefGoogle Scholar
  30. 30.
    Rees, D. C.; Lipscomb, W. N. Refined Crystal-Structure of the Potato Inhibitor Complex of Carboxypeptidase-A at 2.5-A Resolution. J. Mol. Biol. 1982, 160, 475–498.CrossRefGoogle Scholar
  31. 31.
    Bode, W.; Huber, R. Natural Protein Proteinase-Inhibitors and Their Interaction with Proteinases. Eur. J. Biochem. 1992, 204, 433–451.CrossRefGoogle Scholar
  32. 32.
    Marino-Buslje, C.; Venhudova, G.; Molina, M. A.; Oliva, B.; Jorba, X.; Canals, F.; Avilés, F. X.; Querol, E. Contribution of C-Tail Residues of Potato Carboxypeptidase Inhibitor to the Binding to Carboxypeptidase A — A Mutagenesis Analysis. Eur. J. Biochem. 2000, 267, 1502–1509.CrossRefGoogle Scholar
  33. 33.
    Reverter, D.; Vendrell, J.; Canals, F.; Horstmann, J.; Avilés, F. X.; Fritz, H.; Sommerhoff, C. P. A Carboxypeptidase Inhibitor from the Medical Leech Hirudo medicinalis- Isolation, Sequence Analysis, cDNA Cloning, Recombinant Expression, and Characterization. J. Biol. Chem. 1998, 273, 32927–32933.CrossRefGoogle Scholar
  34. 34.
    Frank, M.; Mears, C. A.; Labov, S. E.; Benner, W. H.; Horn, D.; Jaklevic, J. M.; Barfknecht, A. T. High-Efficiency Detection of 66000 Da Protein Molecules Using a Cryogenic Detector in a Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometer. Rapid Commun. Mass Spectrom. 1996, 10, 1946–1950.CrossRefGoogle Scholar
  35. 35.
    Twerenbold, D.; Gerber, D.; Gritti, D.; Gonin, Y.; Netuschill, A.; Rossel, F.; Schenker, D.; Vuilleumier, J. L. Single Molecule Detector for Mass Spectrometry with Mass Independent Detection Efficiency. Proteomics 2001, 1, 66–69.CrossRefGoogle Scholar
  36. 36.
    Yanes, O.; Nazabal, A.; Wenzel, R.; Zenobi, R.; Avilés, F. X. Detection of Noncovalent Complexes in Biological Samples by Intensity Fading and High-Mass Detection Maldi-TOF Mass Spectrometry. J. Proteom. Res. 2006, 5, 2711–2719.CrossRefGoogle Scholar
  37. 37.
    Kiselar, J. G.; Downard, K. M. Preservation and Detection of Specific Antibody-Peptide Complexes by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 746–750.CrossRefGoogle Scholar
  38. 38.
    von Seggern, C. E.; Cotter, R. J. Fragmentation Studies of Noncovalent Sugar-Sugar Complexes by Infrared Atmospheric Pressure Maldi. J. Am. Soc. Mass Spectrom. 2003, 14, 1158–1165.CrossRefGoogle Scholar
  39. 39.
    von Seggern, C. E.; Cotter, R. J. Study of Peptide-Sugar Non-Covalent Complexes by Infrared Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization. J. Mass Spectrom. 2004, 39, 736–742.CrossRefGoogle Scholar
  40. 40.
    Kellersberger, K. A.; Tan, P. V.; Laiko, V. V.; Doroshenko, V. M.; Fabris, D. Atmospheric Pressure Maldi-Fourier Transform Mass Spectrometry. Anal. Chem. 2004, 76, 3930–3934.CrossRefGoogle Scholar
  41. 41.
    Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Yakshin, M. A.; Prasad, C. R.; Lee, H. S.; Doroshenko, V. M. Desorption/Ionization of Biomolecules from Aqueous Solutions at Atmospheric Pressure Using an Infrared Laser at 3 mu m. J. Am. Soc. Mass Spectrom. 2002, 13, 354–361.CrossRefGoogle Scholar
  42. 42.
    Deng, G.; Sanyal, G. Applications of Mass Spectrometry in Early Stages of Target Based Drug Discovery. J. Pharm. Biomed. Anal. 2006, 40, 528–538.CrossRefGoogle Scholar
  43. 43.
    Li, Q.; Ricardo, A.; Benner, S. A.; Winefordner, J. D.; Powell, D. H. Desorption/Ionization on Porous Silicon Mass Spectrometry Studies on Pentose-Borate Complexes. Anal. Chem. 2005, 77, 4503–4508.CrossRefGoogle Scholar
  44. 44.
    Go, E. P.; Apon, J. V.; Luo, G.; Saghatelian, A.; Daniels, R. H.; Sahi, V.; Dubrow, R.; Cravatt, B. F.; Vertes, A.; Siuzdak, G. Desorption/Ionization on Silicon Nanowires. Anal. Chem. 2005, 77, 1641–1646.CrossRefGoogle Scholar
  45. 45.
    Nazabal, A.; Wenzel, R. J.; Zenobi, R. Immunoassays with Direct Mass Spectrometric Detection. Anal. Chem. 2006, 78, 3562–3570.CrossRefGoogle Scholar
  46. 46.
    Wenzel, R. J.; Matter, U.; Schultheis, L.; Zenobi, R. Analysis of Megadalton Ions Using Cryodetection Maldi Time-of-Flight Mass Spectrometry. Anal. Chem. 2005, 77, 4329–4337.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  1. 1.Institut de Biotecnologia i de Biomedicina, and Departament de BioquimicaUniversitat Autònoma de BarcelonaBellaterra (Barcelona)Spain
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark

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