Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution

Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including alpha-helices and beta-sheets. Many native peptides and proteins are predominately alpha-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of alpha-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of alpha-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of alpha-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, Delta HDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of alpha-helix content at a particular Delta HDX, which is indicative of the state of protein folding.