title: Semi-automated screen for global protein conformational changes in solution by ion mobility spectrometry-massspectrometry combined with size-exclusion chromatography and differential hydrogen-deuterium exchange.
abstract: Development of methodologies for studying protein higher-order structure in solution helps to establish a better understanding of the intrinsic link between protein conformational structure and biological function and activity. The goal of this study was to demonstrate a simultaneous screening approach for global protein conformational changes in solution through the combination of ion mobility spectrometry-mass spectrometry (IMS-MS) with differential hydrogen-deuterium exchange (ΔHDX) on the size-exclusion chromatography (SEC) platform in a single on-line workflow. A semi-automated experimental setup based on the use of SEC on-column conditions allowed for tracking of protein conformational changes in solution as a function of acetonitrile concentration. In this setup, the SEC protein elution data was complemented by the ΔHDX profile which showed global protein conformational changes as a difference in the number of deuterons exchanged to protons. The ΔHDX data, in turn, was complemented by the changes in the drift time by IMS-MS. All three orthogonal techniques were applied for studying global higher-order structure of the proteins ubiquitin, cytochrome c and myoglobin, in solution simultaneously. The described approach allows for the use of a crude sample (or mixture of proteins) and could be suitable for rapid comparison of protein batch-to-batch higher-order structure or for optimizing conditions for enzymatic reactions.
authors.affiliation: Merck & Co., Inc., MRL, Analytical Research & Development, Rahway, NJ 07065, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Pierson
authors.affiliation: Merck & Co., Inc., MRL, Analytical Research & Development, Rahway, NJ 07065, USA. Electronic address: alexey.makarov@merck.com.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Makarov
authors.affiliation: Merck & Co., Inc., MRL, Analytical Research & Development, Rahway, NJ 07065, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Strulson
authors.affiliation: Merck & Co., Inc., MRL, Analytical Research & Development, Rahway, NJ 07065, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Merck & Co., Inc., MRL, Analytical Research & Development, Rahway, NJ 07065, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Mao
mesh_terms: D000097:Acetonitriles; D001331:Automation / Q000379:methods; D002850:Chromatography, Gel; D045304:Cytochromes c / Q000737:chemistry; D003903:Deuterium; D041961:Deuterium Exchange Measurement; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D012996:Solutions; D013057:Spectrum Analysis; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D007477:Ions; D009211:Myoglobin; D011522:Protons; D012996:Solutions; D025801:Ubiquitin; D045304:Cytochromes c; D003903:Deuterium; C032159:acetonitrile
keywords: Global conformational change; HDX; IMS; Protein folding; SEC