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.Hydroxyl radical footprinting (HRF)
1 is valuable for assessing the structure of macromolecules. Single nucleotide resolution data enabled by the similar reactivity of the OH radical with each and every backbone position has helped solve important problems in the nucleic acids field, such as understanding RNA folding and ribosome assembly (
1–
5). Applications of HRF to probe protein structure are a subset of a family of structural MS approaches, including the use of reversible deuterium labeling or irreversible covalent labeling, including labeling with OH radicals (
6–
13). Hydrogen-deuterium exchange MS (HDX-MS) is particularly suited to measure secondary and tertiary structure stability through backbone exchange, whereas HRF-MS has been effective at measuring the relative solvent accessibility of specific amino acid side chains mediated by intramolecular tertiary and intermolecular quaternary structure interactions. Hydroxyl radicals can be generated by a variety of methods in each case the chemistry has been shown to be quite similar and the radicals react with side chains of surface residues resulting in well characterized oxidation products (
7,
10,
11). As up to 18 side chains are potential probes, the overall protein coverage and resolution of the method is theoretically high.Both HDX-MS and HRF-MS utilize a “bottom-up” proteomics approach where proteins are digested to peptide states after labeling, and mass shifts of the resultant peptides are read-out to pinpoint sites of conformational change. Although this usually can provide 90% or more coverage across the entire protein length, in fact the structural resolution is limited as the size of the peptide fragments and the data report the average behavior of the individual residues across the entire peptide, which are typically in the range of five–20 residues (
14). MS2 based quantification is in principle a general solution to the problem of increasing structural resolution, and has been attempted for HDX-MS, but the scrambling of the labels in the gas phase has been difficult to overcome using collision induced dissociation (
15,
16). Alternative approaches for HDX-MS site localization, like electron transfer dissociation to achieve single residue resolution have potential promise but are typically limited to larger peptides that can access higher charge states easily (
17,
18). MS2 strategies to enhance the resolution for covalent labeling experiments have been attempted with some success, as scrambling is not a limitation in covalent labeling experiments (
7,
19–
21). On the other hand, MS1 based strategies to enhance structural resolution for both HDX and covalent labeling approaches using overlapping protease fragments are also a promising route to providing subpeptide resolution in many cases (
7,
20–
27).In this work, we present a coupled set of high-throughput experimental and computational approaches to extend previous MS2 based HRF-MS strategies and provide a quantitative topographical structure assessment for proteins at the individual side chain level. The combined approach permits quantification of modifications through examination of a tandem-ion based ladder of peptide fragments and combining the ion abundances from both MS1 and MS2 quantification. The high-resolution information is transformed using the knowledge of the relative reactivity of side chains to predict side-chain surface area for the structurally well-characterized Ca
2+ bound form of Calmodulin (CaM). In addition, we explored a statistical approach using random forest regression methods to predict solvent accessible surface area at the residue level. Overall, these studies provide a novel approach to provide high-resolution single-residue surface accessibility data with at least eightfold higher spatial resolution than peptide based measures for accurate protein topography predictions.
相似文献