title: Structural Characterization of Missense Mutations Using High Resolution Mass Spectrometry: A Case Study of the Parkinson's-Related Protein, DJ-1.
abstract: Missense mutations that lead to the expression of mutant proteins carrying single amino acid substitutions are the cause of numerous diseases. Unlike gene lesions, insertions, deletions, nonsense mutations, or modified RNA splicing, which affect the length of a polypeptide, or determine whether a polypeptide is translated at all, missense mutations exert more subtle effects on protein structure, which are often difficult to evaluate. Here, we took advantage of the spectral resolution afforded by the EMR Orbitrap platform, to generate a mass spectrometry-based approach relying on simultaneous measurements of the wild-type protein and the missense variants. This approach not only considerably shortens the analysis time due to the concurrent acquisition but, more importantly, enables direct comparisons between the wild-type protein and the variants, allowing identification of even subtle structural changes. We demonstrate our approach using the Parkinson's-associated protein, DJ-1. Together with the wild-type protein, we examined two missense mutants, DJ-1A104T and DJ-1D149A, which lead to early-onset familial Parkinson's disease. Gas-phase, thermal, and chemical stability assays indicate clear alterations in the conformational stability of the two mutants: the structural stability of DJ-1D149A is reduced, whereas that of DJ-1A104T is enhanced. Overall, we anticipate that the methodology presented here will be applicable to numerous other missense mutants, promoting the structural investigations of multiple variants of the same protein. Graphical Abstract ᅟ.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 7610001, Israel.
authors.forename: Gili
authors.initials: G
authors.lastname: Ben-Nissan
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 7610001, Israel.
authors.forename: Almog
authors.initials: A
authors.lastname: Chotiner
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 7610001, Israel.
authors.forename: Mark
authors.initials: M
authors.lastname: Tarnavsky
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 7610001, Israel. michal.sharon@weizmann.ac.il.
authors.forename: Michal
authors.initials: M
authors.lastname: Sharon
mesh_terms: D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D020125:Mutation, Missense; D015513:Oncogene Proteins; D010300:Parkinson Disease; D000071617:Protein Deglycase DJ-1 / Q000737:chemistry* / Q000235:genetics
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D015513:Oncogene Proteins; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1
keywords: DJ-1; Missense mutations; Native mass spectrometry; Orbitrap EMR mass spectrometer; Parkinson’s disease