title: Does deamidation cause protein unfolding? A top-down tandem mass spectrometry study.
abstract: Deamidation is a nonenzymatic post-translational modification of asparagine to aspartic acid or glutamine to glutamic acid, converting an uncharged amino acid to a negatively charged residue. It is plausible that deamidation of asparagine and glutamine residues would result in disruption of a proteins' hydrogen bonding network and thus lead to protein unfolding. To test this hypothesis Calmodulin and B2M were deamidated and analyzed using tandem mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The gas phase hydrogen bonding networks of deamidated and nondeamidated protein isoforms were probed by varying the infra-red multi-photon dissociation laser power in a linear fashion and plotting the resulting electron capture dissociation fragment intensities as a melting curve at each amino acid residue. Analysis of the unfolding maps highlighted increased fragmentation at lower laser powers localized around heavily deamidated regions of the proteins. In addition fragment intensities were decreased across the rest of the proteins which we propose is because of the formation of salt-bridges strengthening the intramolecular interactions of the central regions. These results were supported by a computational flexibility analysis of the mutant and unmodified proteins, which would suggest that deamidation can affect the global structure of a protein via modification of the hydrogen bonding network near the deamidation site and that top down FTICR-MS is an appropriate technique for studying protein folding.
authors: Department of Chemistry, University of Warwick, Coventry, United Kingdom.
authors: Andrew J
authors: AJ
authors: Soulby
authors: Jack W
authors: JW
authors: Heal
authors: Mark P
authors: MP
authors: Barrow
authors: Rudolf A
authors: RA
authors: Roemer
authors: Peter B
authors: PB
authors: O'Connor
mesh_terms: D000577:Amides / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D006860:Hydrogen Bonding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D058767:Protein Unfolding; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000577:Amides; D002147:Calmodulin; D001613:beta 2-Microglobulin; D005973:Glutamine; D001216:Asparagine
keywords: deamidation; mass spectrometry; protein; unfolding
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