Molecular recognition specificity and bioaffinity quantification in biopolymer interaction of anti 3-nitrotyrosine antibody revealed by SAW-ESI-MS and PLIMSTEX dilutions strategy

摘要

Tyrosine nitration in proteins plays an important role in many diseases such as Alzheimer's disease, Parkinson, atherosclerosis and bronchio-alveolar diseases. Molecular information about specificity, reactivity and the epitope recognised by anti-nitrotyrosyl antibodies is essential for analytical use and quality assurance of antibodies, especially in the light of their extensive use in diagnostic applications. The aim of the present study is to characterize the protein-ligand interaction between nitrated peptides and specific m anti 3-nitrotyrosine antibody by MS using two methods which were recently developed (a) direct coupling of surface acoustic wave (SAW) with electrospray mass spectrometry (SAW-ESI-MS) and (b) PLIMSTEX dilustions strategy (dPLIMSTEX). Online coupling of an S-Sens K5 surface acoustic wave biosensor with ESI-MS (SAW-ESI-MS) enables the directly molecular identification of protein-protein interactions involved in complex macromolecular assemblies such as antibody-antigen complexes; the characterisation of binding affinity by mass spectrometry allows the identification of unknown binding partners. Monitoring of molecular interaction kinetics between antibodies and peptides or proteins is provided by SAW, and analysed by coupling with ESI- MS. Under the flow of the biosensor the affinity-bound material is directed eluted into a guard column which is coupled to an injection unit. The guard column provides an efficient in-situ concentration and desalting for MS analysis. The new method dPLIMSTEX was applied for characterization of the affinity binding between antibody against nitrated tyrosine and nitrated tyrosine peptides or proteins, conformational changes, binding stoichiometry and binding constant.