Over-expression 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 micro-heterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top down 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 micro-heterogeneities that were hitherto only accessible with FTICR-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both wt and K496C variant of the recombinant X domain (XD, aa 459-507) of the measles virus phosphoprotein expressed in E. coli. Using top down MSMS combined to TWIM, we show that XD samples occasionally exhibit a micro-heterogeneity 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, an MTSL nitroxide probe that was grafted on the K496C XD variant was shown to undergo oxidation and/or protonation in the ESI source leading to artifactual mass increases.
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