Quantitative protein topography analysis and high-resolution structure prediction using hydroxyl radical labeling and tandem-ion mass spectrometry (MS)

Abstract

Hydroxyl radical footprinting based MS for protein structure assessment has the goal of understanding ligand induced conformational changes and macromolecular interactions, for example, protein tertiary and quaternary structure, but the structural resolution provided by typical peptide-level quantification is limiting. In this work, we present experimental strategies using tandem-MS fragmentation to increase the spatial resolution of the technique to the single residue level to provide a high precision tool for molecular biophysics research. Overall, in this study we demonstrated an eightfold increase in structural resolution compared with peptide level assessments. In addition, to provide a quantitative analysis of residue based solvent accessibility and protein topography as a basis for high-resolution structure prediction; we illustrate strategies of data transformation using the relative reactivity of side chains as a normalization strategy and predict side-chain surface area from the footprinting data. We tested the methods by examination of Ca(+2)-calmodulin showing highly significant correlations between surface area and side-chain contact predictions for individual side chains and the crystal structure. Tandem ion based hydroxyl radical footprinting-MS provides quantitative high-resolution protein topology information in solution that can fill existing gaps in structure determination for large proteins and macromolecular complexes.

Fig. 1.

Workflow steps for A, Setting up the MS experimental method B, Data analysis. DR: dose response, MS: mass spectrometry, m/z: mass-to-charge ratio, RC: rate constant, RT: retention time, SIC: selected ion chromatogram.

Fig. 2.

A, UPLC separated single ion chromatograms of doubly charged, unmodified (solid) and +16 modified (dotted) forms of Calmodulin peptide 1–13 showing multiple isomers of the +16 oxidation. Most peaks are comprised of multiple sites of oxidation as marked. B–C, Combo selected ion chromatograms (SICs) of fragment ions from peptide 1–13. The blue line indicates the unoxidized ion, red plot shows extraction of the +16 oxidized form of the fragment ion (e.g. y4 + 16), and the green plot depicts the extraction of the unlabeled ion (e.g. y4) from the +16 oxidized form of the peptide. Interestingly, the red and the green plots get reversed in both the complementary product ion pairs b9/y4 and b8/y5. D, Extent of +16 oxidation increases with the increase in the number of constituent residues of each ion, showing the measurement of subtle oxidation changes. Complementary product ion pairs are shown concurrently.

Fig. 3.

Dose response plots of CaM peptides: A, 1–13, b-series; B, 1–13, y-series; C, 14–21, b-series; D, 14–21, y-series. The dotted line marked by T shows the total unoxidized peptide.

Fig. 4.

A, Relation between fractional SASA based on crystal structure and prediction using Random Forest regression. Rate constants from b- and y-ions showed a Pearson's correlation coefficient of 0.995, and were averaged to make predictions for each residue. The diagonal line is shown in blue, indicating the ideal behavior. The Pearson's correlation coefficient between the two values across 40 residues is 0.79 (p value = 2.3 × 10⁻⁹). B, Two views of the crystal structure of calmodulin mapped with predicted solvent accessibility. High protection is shown in blue regions, purple regions show medium protection, whereas red regions show high solvent accessibility. Ca²⁺ ions are shown by white spheres.