Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein

• Published in: Journal of the American Chemical Society
• Main Authors: Pan, Jingxi, Han, Jun, Borchers, Christoph H., Konermann, Lars
• Format: Article in Journal/Newspaper
• Language: unknown
• Published: Scholarship@Western 2009
• Subjects: Amino Acid Sequence; Animals; Apoproteins; Cattle; Deuterium Exchange Measurement; Electrons; Hydrogen Bonding; Hydrophobicity; Mass Spectrometry; Models; Molecular; Molecular Sequence Data; Molecular Weight; Myoglobin; Protein Folding; Protein Structure; Secondary; Chemistry; Medical Biochemistry; Holo; Sperm whale
• Online Access: https://ir.lib.uwo.ca/chempub/9; https://doi.org/10.1021/ja904379w; http://pubs.acs.org/doi/abs/10.1021/ja904379w

Amide H/D exchange (HDX) mass spectrometry (MS) is widely used for protein structural studies. Traditionally, this technique involves protein labeling in D(2)O, followed by acid quenching, proteolytic digestion, and analysis of peptide deuteration levels by HPLC/MS. There is great interest in the development of alternative HDX approaches involving the top-down fragmentation of electrosprayed protein ions, instead of relying on enzymatic cleavage and solution-phase separations. A number of recent studies have demonstrated that electron capture dissociation (ECD) results in fragmentation of gaseous protein ions with little or no H/D scrambling. However, the successful application of this approach for in-depth protein conformational studies has not yet been demonstrated. The current work uses horse myoglobin as a model system for assessing the suitability of HDX-MS with top-down ECD for experiments of this kind. It is found that ECD can pinpoint the locations of protected amides with an average resolution of less than two residues for this 17 kDa protein. Native holo-myoglobin (hMb) shows considerable protection from exchange in all of its helices, whereas loops are extensively deuterated. Fraying is observable at some helix termini. Removal of the prosthetic heme group from hMb produces apo-myoglobin (aMb). Both hMb and aMb share virtually the same HDX protection pattern in helices A-E, whereas helix F is unfolded in aMb. In addition, destabilization is evident for some residues close to the beginning of helix G, the end of helix H, and the C-terminus of the protein. The structural changes reported herein are largely consistent with earlier NMR data for sperm whale myoglobin, although small differences between the two systems are evident. Our findings demonstrate that the level of structural information obtainable with top-down ECD for small to medium-sized proteins considerably surpasses that of traditional HDX-MS experiments, while at the same time greatly reducing undesired amide back exchange.