Mass spectrometry-based biomolecular recognition and response factor investigations using electrospray ionization.

摘要

Integrins are transmembrane proteins found in almost all cells. They play vital roles in many biological processes, such as angiogenesis and signal transduction, through their interactions with different extracellular matrix ligands. These interactions are fostered by forming noncovalent bonds with the ligands. Integrin involvement in angiogenic cancer metastasis has sparked the interest of many researchers in the field of oncology. Many cutting edge research works are now focused on studying the noncovalent interactions of integrins with potential drug compounds formulated from peptide mimics and bearing the putative RGD amino acid sequence through which integrins are known to bind their ligands. Most of the published works on the study of noncovalent interactions of integrins with their ligands have been carried out using traditional techniques such as X-Ray crystallography, NMR, and immunoassay development. The introduction of ESI-MS to the list of viable techniques for probing the integrin-RGD noncovalent interactions is a logical and much needed approach due to its versatility and high throughput capability compared to the more traditional techniques.;The experiments described in this dissertation serve to establish, for the first time, the amenability of ESI-MS for assessing integrin-RGD binding affinity using only peptide fragments that represent the binding region of the intact integrin protein molecule. Using information from crystallographic data obtained from the literature about the amino acid sequence of the integrin binding domain, peptide fragments were synthesized having these amino acid sequences and tested for affinity to different short peptide ligands. Important considerations regarding analyte response factors during ESI-MS process, and how these impact binding affinity determination in ESI-MS, were also addressed experimentally and in conjunction with statistical tools.;Three major challenges are noted with this new approach. The first has to do with marked difference between the conformations of the peptide fragments employed compared to that of the tertiary structure of the integrin molecule. Also, the binding constants in these experimental conditions may differ from actual physiological conditions. And lastly is the question of how the response factors of host, guest, and complex affect the measured binding affinities.