Complexes between recombinant intracellular carriers of vitamin A and their specific ligands investigated by electrospray-mass spectrometry.

ABSTRACT

The intracellular carriers of vitamin A, cellular retinol-binding protein type I, cellular retinol-binding protein type II and cellular retinoic acid-binding protein type I are members of the intracellular lipid-binding proteins family, in which the ligand-binding cavity is located in the interior of a barrel-like structure. The dissociation constants of the specific complexes in water solutions around neutrality are very low (in the 0.1 to 10 nM range). Because of their high stability, they represent ideal systems to verify the adequacy of electrospray ionization-mass spectrometry in the analysis of non-covalent protein-ligand complexes. The electrospray interface parameters were varied to detect the presence of species not present in solution but generated as artefacts during transfer of complexes from the condensed state to the gas-phase. The results clearly indicate that mass-spectrometry data reflect the situation present in solution only if the electrospray conditions are carefully selected. In particular, the values of cone voltage and temperature compatible with persistence of the complexes in the gas phase were determined for each vitamin A carrier. Lack of correlation between complex stability in solution and in the gas phase is attributable to the specific and differential effects of the two environments on protein conformation and ligand-protein interactions.