The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclo tron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the milli molar range, such as pk trisaccharide (globotriaoside) complexed with the S higa-like toxin (SLT) of pathogenic E, coli, It is further demonstrated tha t nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multim eric proteins, such as the SLTs, At increasing trisaccharide:protein ratios it was shown that the B, toxin subunit complexes with 5 pk trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound pk's af the five binding sites, it was possible to establish association c onstants for each of the five sites and to confirm that binding occurs nonc ooperatively, the association constants for each site are identical and tha t compared to site 1, site 2 exhibits a tenfold higher affinity for the glo botriaoside synthetic ligand I. The facile identification of the occupancy of binding sites represents information that is not readily available by ot her techniques. This sensitive and rapid estimation of association constant s for protein-ligand complexes, which are free of unpredictable secondary e ffects that plague enzyme linked assays, is likely to find wide application .