10.1021/ac010713f.s001
Andras Dobo
Andras
Dobo
Igor A. Kaltashov
Igor A.
Kaltashov
Detection of Multiple Protein Conformational
Ensembles in Solution via Deconvolution of
Charge-State Distributions in ESI MS
American Chemical Society
2001
CRABP
Average charge state
charge state n
ESI MS Monitoring
solution
protein ion charge scale n
basis functions B i
i.e
pH
Multiple Protein Conformational Ensembles
retinoic acid binding protein
analysis
coefficients b i
basis function
2001-09-15 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Detection_of_Multiple_Protein_Conformational_Ensembles_in_Solution_via_Deconvolution_of_Charge-State_Distributions_in_ESI_MS/3582735
Monitoring the changes in charge-state distributions of
protein ions in electrospray ionization (ESI) mass spectra
has become one of the commonly accepted tools to detect
large-scale conformational changes of proteins in solution.
However, these experiments produce only qualitative, low-resolution information. Our goal is to develop a procedure
that would produce quantitative data on protein conformational isomers coexisting in solution at equilibrium. To
that end, we have examined the evolution of positive ion
charge-state distributions in the ESI spectra of two model
proteins, α-helical myoglobin (Mb) and β-sheet cellular
retinoic acid binding protein I (CRABP I), as a function
of solution pH. A detailed analysis of the charge-state
distributions over a wide range of pH (2.6−8.5) suggests
that each spectrum (i.e., relative ion abundance <i>I</i> vs its
charge state <i>n</i>) can be approximated as a linear combination of a limited number of basis functions <i>B</i><i><sub>i</sub></i>(<i>n</i>), i.e. <i>I</i>(<i>n</i>)
= ∑<i>b</i><i><sub>i</sub></i><i>B</i><i><sub>i</sub></i>(<i>n</i>). These basis functions (approximated as
normal, or Gaussian, distributions) are not significantly
affected by the pH variations; however, their relative
intensities (coefficients <i>b</i><i><sub>i</sub></i>) exhibit strong pH dependence
giving rise to complicated overall charge-state distributions. Analysis of the experimental data, aided by the vast
existing body of knowledge of Mb and CRABP I conformational properties (both structure and dynamics) leads
to a conclusion that each basis function in fact represents
a single conformational isomer. Average charge state
corresponding to each basis function (e.g., position of the
maximum of <i>B</i><i><sub>i</sub></i>(<i>n</i>) on the protein ion charge scale <i>n</i>)
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 heterogeneity. Overall, this technique is suitable for analysis of complex mixtures of
protein conformational isomers in solution and complements existing experimental methods that are used to
study macromolecular dynamics by characterizing protein
shape in solution (e.g., scattering techniques).