LemonCatcher Acidic Pull-Down Enables Selective In-Cell Hydrogen-Deuterium Exchange Mass Spectrometry
Hammerschmid, D.; Ehsani, M.; Keeble, A. H.; Russell Lewis, B.; Calvaresi, V.; Heatley, P.; Zhu, D.; Hayward, H.; Struwe, W. B.; Booth, P. J.; Howarth, M. R.; Reading, E.
Show abstract
Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.
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