title: Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.
abstract: Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Cummins
authors.affiliation: Analytical Technologies Department, Centro de Investigación Lilly, SA , Avenida de la Industria 30, 28108 Alcobendas, Spain.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Espada
authors.affiliation: The Scripps Research Institute , Department of Molecular Therapeutics, 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Novick
authors.affiliation: Analytical Technologies Department, Centro de Investigación Lilly, SA , Avenida de la Industria 30, 28108 Alcobendas, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Molina-Martin
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Stites
authors.affiliation: Analytical Technologies Department, Centro de Investigación Lilly, SA , Avenida de la Industria 30, 28108 Alcobendas, Spain.
authors.forename: Juan Felix
authors.initials: JF
authors.lastname: Espinosa
authors.affiliation: Analytical Technologies Department, Centro de Investigación Lilly, SA , Avenida de la Industria 30, 28108 Alcobendas, Spain.
authors.forename: Howard
authors.initials: H
authors.lastname: Broughton
authors.affiliation: The Scripps Research Institute , Department of Molecular Therapeutics, 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Devrishi
authors.initials: D
authors.lastname: Goswami
authors.affiliation: The Scripps Research institute, Informatics Core, 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Pascal
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Dodge
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Chalmers
authors.affiliation: The Scripps Research Institute , Department of Molecular Therapeutics, 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Griffin
mesh_terms: D000704:Analysis of Variance; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003903:Deuterium / Q000032:analysis; D041961:Deuterium Exchange Measurement / Q000295:instrumentation / Q000379:methods*; D006859:Hydrogen / Q000032:analysis; D008024:Ligands; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000737:chemistry; D018167:Receptors, Calcitriol / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D008024:Ligands; D010455:Peptides; D011506:Proteins; D018167:Receptors, Calcitriol; D006859:Hydrogen; D003903:Deuterium