Neutron and ion beams in biological research -: On the scattering length density of proteins in H2O/D2O:: Determination of H-D exchange using ES+ I-MS

Neutron reflectometry is an excellent technique to determine the structure of a protein layer adsorbed at a solid interface. The use of contrast-variation makes it possible to highlight the adsorbed protein layer. The neutron scattering lengths of D and H have opposite signs. By choosing the H2O/D2O ratio, the scattering length density can be made equal to that of the adsorbing substrate. To determine the scattering length density of a protein, both its volume and its scattering length in solution is needed. To calculate the scattering length, the H-D exchange of the labile protons of the protein should be taken into account. For monitoring the H-D exchange, electrospray ionization mass spectroscopy (ES+I-MS) was applied. We show that ES+I-MS is the appropriate technique for quantitative analysis of the H-D exchange. We compare the experimental results for the exchange in lysozyme and beta-casein with theoretical calculations.