Detection of multiple protein conformational ensembles in solution via deconvolution of charge-state distributions in ESI MS

Monitoring the changes in charge-state distributions of protein ions in ele ctrospray ionization (ESI) mass spectra has become one of the commonly acce pted tools to detect large-scale conformational changes of proteins in solu tion. However, these experiments produce only qualitative, low-resolution i nformation. Our goal is to develop a procedure that would produce quantitat ive data on protein conformational isomers coexisting in solution at equili brium. To that end, we have examined the evolution of positive ion charge-s tate distributions in the ESI spectra of two model proteins, alpha -helical myoglobin (Mb) and beta -sheet cellular retinoic acid binding protein I (C RABP I), as a function of solution pH. A detailed analysis of the charge-st ate distributions over a wide range of pH (2.6-8.5) suggests that each spec trum (i.e., relative ion abundance I vs its charge state n) can be approxim ated as a linear combination of a limited number of basis functions B-i(n), i.e. I(n) = Sigmab(i)B(i)(n). These basis functions (approximated as norma l, or Gaussian, distributions) are not significantly affected by the pH var iations; however, their relative intensities (coefficients b(i)) exhibit st rong pH dependence giving rise to complicated overall charge-state distribu tions. Analysis of the experimental data, aided by the vast existing body o f knowledge of Mb and CRABP I conformational properties (both structure and dynamics) leads to a conclusion that each basis function in fact represent s a single conformational isomer. Average charge state corresponding to eac h basis function (e.g., position of the maximum of B-i(n) on the protein io n charge scale n) characterizes the conformer's overall shape (most likely, projected surface area). The width of each basis function (i.e., standard deviation of the normal distribution) represents the conformer's heterogene ity. Overall, this technique is suitable for analysis of complex mixtures o f protein conformational isomers in solution and complements existing exper imental methods that are used to study macromolecular dynamics by character izing protein shape in solution (e.g., scattering techniques).