title: Dividing to unveil protein microheterogeneities: traveling wave ion mobility study.
abstract: Overexpression of a protein in a foreign host is often the only route toward an exhaustive characterization, especially when purification from the natural source(s) is hardly achievable. The key issue in these studies relies on quality control of the purified recombinant protein to precisely determining its identity as well as any undesirable microheterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top-down tandem mass spectrometry (MSMS) requires the use of highly accurate and highly resolved experiments to reveal subtle sequence modifications. In the present study, the top-down MSMS approach combined with traveling wave ion mobility (TWIM) separation was evaluated for its ability to achieve high sequence coverage and to reveal subtle microheterogeneities that were hitherto only accessible with Fourier-transform ion cyclotron resonance-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both the wild type and K496C variant of the recombinant X domain (XD; aa's 459-507) of the measles virus phosphoprotein expressed in  Escherichia coli . Using top-down MSMS combined with TWIM, we show that XD samples occasionally exhibit a microheterogeneity that could not be anticipated from the nucleotide sequence of the encoding constructs and that likely reflects a genetic drift, neutral or not, occurring during expression. In addition, a 1-oxyl-2,2,5,5-tetramethyl-δ3-pyrroline-3-methyl methanethiosulfonate nitroxide probe that was grafted onto the K496C XD variant was shown to undergo oxidation and/or protonation in the electrospray ionization source, leading to artifactual mass increases.
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, Equipe de Protéomique Fonctionnelle et Dynamique, UPR 9036-CNRS, 31 Chemin Joseph Aiguier, 13420 Marseille Cedex, France. fhalgand@ifr88.cnrs-mrs.fr
authors.forename: F
authors.initials: F
authors.lastname: Halgand
authors.forename: Johnny
authors.initials: J
authors.lastname: Habchi
authors.forename: Laetitia
authors.initials: L
authors.lastname: Cravello
authors.forename: Marlène
authors.initials: M
authors.lastname: Martinho
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.forename: Sonia
authors.initials: S
authors.lastname: Longhi
mesh_terms: D008459:Measles virus / Q000235:genetics; D008958:Models, Molecular; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D011994:Recombinant Proteins