Top-Down Structural Analysis of Post-Translationally Modified Proteins by FTICR-MS with H/D Exchange and Electron Capture Dissociation.

Proteomics. 2013 Mar;13(6):974-81. doi: 10.1002/pmic.201200246. Epub 2013 Feb 15.
Pan J, Borchers CH.

High-resolution structural characterization of post-translationally-modified proteins represents a challenge for traditional structural biology methods such as crystallography and NMR. In this study, we have used top-down hydrogen/deuterium exchange mass spectrometry (HDX-MS) with precursor ion selection and electron capture dissociation (ECD) to determine the impact of oxidative modification on calmodulin (CaM) at an average resolution of 2.5 residues, with complete sequence coverage. The amide deuteration status of native CaM determined by this method correlates well with previously reported crystallographic and NMR data. In contrast, methionine oxidation caused almost complete deuteration of all residues in the protein in 10 seconds. The oxidative-modification-induced secondary and tertiary structure loss can be largely recovered upon calcium ligation, which also resulted in a substantial increase of amide protection in three of the four calcium-binding loops in oxidatively-modified CaM (CaM(ox) ). However, the structure of α-helix VI is not restored by cofactor binding. These results are discussed in terms of different target binding and activation capabilities displayed by CaM and CaM(ox) . The isoform-specific top-down HDX structural analysis strategy demonstrated in this study should be readily applicable to other oxidatively-modified proteins and other types of PTMs, and may help decipher the structure and function of specific protein isoforms.