Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure

Specific amino acid footprinting mass spectrometry (MS)
is an increasingly
utilized method for elucidating protein higher order structure (HOS).
It does this by adding to certain amino acid residues a mass tag,
whose reaction extent depends on solvent accessibility and microenvironment
of the protein. Unlike reactive free radicals and carbenes, these
specific footprinters react slower than protein unfolding. Thus, their
footprinting, under certain conditions, provokes structural changes
to the protein, leading to labeling on non-native structures. It is
critical to establish conditions (i.e., reagent concentrations, time
of reaction) to ensure that the structure of the protein following
footprinting remains native. Here, we compare the efficacy of five
methods in assessing protein HOS following footprinting at the intact
protein level and then further localize the perturbation at the peptide
level. Three are MS-based methods that provide dose–response
plot analysis, evaluation of Poisson distributions of precursor and
products, and determination of the average number of modifications.
These MS-based methods reliably and effectively indicate HOS perturbation
at the intact protein level, whereas spectroscopic methods (circular
dichroism (CD) and dynamic light scattering (DLS)) are less sensitive
in monitoring subtle HOS perturbation caused by footprinting. Evaluation
of HOS at the peptide level indicates regions that are sensitive to
localized perturbations. Peptide-level analysis also provides higher
resolution of the HOS perturbation, and we recommend using it for
future footprinting studies. Overall, this work shows conclusive evidence
for HOS perturbation caused by footprinting. Implementation of quality
control workflows can identify conditions to avoid the perturbation,
for footprinting, allowing accurate and reliable identification of
protein structural changes that accompany, for example, ligand interactions,
mutations, and changes in solution environment.