Use of Raman spectroscopy and size-exclusion chromatography coupled with HDX-MS spectroscopy for studying conformational changes of small proteins in solution.

Protein-based drugs are a relatively new paradigm in modern therapeutics. These large molecule drugs often have much higher specificity and selectivity compared to small molecules that have been used in therapeutics for centuries. However, there are many analytical challenges associated with drug discovery and development of these new modalities. One of these analytical challenges concerns fast and robust assessment of peptides or small protein conformational structures in solution. In this study, we report a novel analytical approach that is based on Raman spectroscopy (RS) and size exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) for probing conformational structures of proteins in solution. Specifically, we demonstrate that RS and SEC-HDX-MS can be used to probe temperature-induced changes in ubiquitin and insulin. We also show that a combination of these techniques provides a more comprehensive analysis and comparison of peptide or small protein conformational structures than by any one technique. Our results demonstrate that RS and SEC-HDX-MS allow for elucidation of sequential transformations in α-helix and ß-sheet content of these proteins. These findings suggest that the proposed approach can be used for a fast investigation of changes in protein or peptide secondary structures under different solution conditions.