Development of an ESI-MS screening method for evaluating binding affinity between integrin fragments and RGD-based peptides

Misjudeen Raji, P. Frycak, M. Beall, M. Sakrout, J. M. Ahn, Y. Bao, D. W. Armstrong, K. A. Schug*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    18 Scopus citations


    Electrospray ionization mass spectrometry (ESI-MS) is used to evaluate binding affinities between integrin fragments and RGD-based peptide ligands. The integrin fragment peptides synthesized and studied were selected based on crystallographic and literature data which highlighted the specific binding pockets for the RGD motif in intact integrin proteins. Relative binding constants were determined by fitting the data obtained from ESI-MS titration experiments to a quadratic equation based on a 1:1 association model. Frontal analysis capillary electrophoresis (FACE) was used as a complementary solution phase technique to measure the absolute binding constants for the same host-guest systems and the results from both techniques were compared. Gas phase collision activated dissociation threshold measurements in an ion trap mass spectrometer also were carried out on the complexes to further validate binding constant measurements. Better correlation was observed between the MS/MS and CE results, than for ESI-MS titration. The ESI-MS titration results showed some correlation; however, it is apparent that there are important methodological shortcomings which need to be addressed. Several factors related to the titration method, ionization efficiencies, enthalpy/entropy contributions during phase transfer, and solvent effects are discussed. This work introduces a first approach to development of a high throughput ESI-MS-based method suitable for screening potential peptide-based drug compounds that target integrins on cancerous cells. It also serves as a cautionary tale to those interested in performing similar types of analyses.

    Original languageEnglish (US)
    Pages (from-to)232-240
    Number of pages9
    JournalInternational Journal of Mass Spectrometry
    Issue number3
    StatePublished - May 1 2007


    • Binding constant
    • Collision threshold
    • Integrin
    • RGD motif
    • Titration

    ASJC Scopus subject areas

    • Physical and Theoretical Chemistry
    • Spectroscopy


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