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Biochemistry Seminar – Alexey Makarov:”Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Screening Approaches for Studying Global Conformational Structures of Peptides/ Proteins in Solution”
February 6 @ 4:00 pm - 5:00 pm
Analytical Chemistry Enabling Technologies from Merck
Title:”Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Screening Approaches for Studying Global Conformational Structures of Peptides/ Proteins in Solution”
Methodologies for studying protein higher-order structure in solution can help to establish a better understanding of the intrinsic link between proteins’ conformational structure and their biological function and activity. In this presentation we will demonstrate the applicability of screening approaches for studying global protein conformational changes in solution. Several screening approaches were surveyed in combination with differential hydrogen-deuterium exchange. We describe an application of chromatography hyphenated techniques based on coupling as a single on-line workflow with differential hydrogen-deuterium exchange (HDX-MS). One technique joins HDX-MS with size-exclusion chromatography (SEC). 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. Another technique utilized ultra-high pressure liquid chromatography (UHPLC) to explore compressibility of the higher order structure of proteins under increasing pressure detected by HDX. It was found that the increase of retention factors upon pressure increase, at constant flow rate and temperature, was based on reduction of the proteins’ molecular molar volume. The change in the proteins’ molecular molar volume was caused by changes in protein folding, as was demonstrated by differential HDX. By modifying pressure during UHPLC separation, it was possible to achieve changes in protein folding, which were manifested as changes in the number of labile protons exchanged to deuterons, or vice-versa.
Location: 108 Biochemistry Building (Bldg#1507)