title: Combining size-exclusion chromatography with differential hydrogen-deuterium exchange to study protein conformational changes.
abstract: Methods for protein characterization are being actively developed based on the growing importance of protein therapies and applications. The goal of this study was to demonstrate the use of size-exclusion chromatography (SEC) in combination with differential hydrogen-deuterium exchange (HDX) to compare protein global conformational changes at different solution conditions. Using chaotropic mobile phase additive, differential HDX was used to detect a number of solvent accessible labile protons of protein on-column at pH and temperature conditions which provided unrestricted intrinsic H/D exchange (all-or-nothing approach). Varying SEC on-column conditions allowed for protein conformational changes to be observed. Temperature and pressure were independently studied with regards to their effect on the proteins' (insulin, cytochrome C, ubiquitin, and myoglobin) conformational changes in the solution. The obtained ΔHDX profiles revealed protein conformational changes in solution under varied conditions manifested as the difference in the number of protons exchanged to deuterons, or vice-versa. The approach described in this manuscript could prove useful for protein batch-to-batch comparisons, for optimization of chemical reactions with enzyme as catalyst or for protein chemical modification reactions.
authors.affiliation: Department of Process & Analytical Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA. Electronic address: alexey.makarov@merck.com.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Makarov
authors.affiliation: Department of Process & Analytical Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: Roy
authors.initials: R
authors.lastname: Helmy
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002850:Chromatography, Gel; D045304:Cytochromes c / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry; D011487:Protein Conformation; D013696:Temperature; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D009211:Myoglobin; D025801:Ubiquitin; D006859:Hydrogen; D045304:Cytochromes c; D003903:Deuterium
keywords: Chaotropic additive; Global conformational changes; HDX; Protein in solution; SEC; UHPLC