Laboratoire de Bioénergétique et Ingénierie des Protéines, Equipe de Protéomique Fonctionnelle et Dynamique, UPR 9036-CNRS, 31 Chemin Joseph Aiguier, 13420 Marseille Cedex, France.
Anal Chem. 2011 Oct 1;83(19):7306-15. doi: 10.1021/ac200994c. Epub 2011 Aug 29.
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.
在异源宿主中过表达蛋白质通常是进行详尽表征的唯一途径，尤其是当从天然来源（s）中纯化几乎不可能时。这些研究中的关键问题在于对纯化的重组蛋白进行质量控制，以准确确定其身份以及任何不理想的微观异质性。虽然标准蛋白质组学方法排除了对修饰的无偏搜索，但自上而下串联质谱（MSMS）的可选技术需要使用高度准确和高分辨率的实验来揭示微妙的序列修饰。在本研究中，评估了自上而下的 MSMS 方法与行进波离子淌度（TWIM）分离相结合的能力，以实现高序列覆盖率并揭示迄今为止仅使用傅里叶变换离子回旋共振-MS 仪器才能获得的微妙微观异质性。本研究通过对在大肠杆菌中表达的麻疹病毒磷酸蛋白的重组 X 结构域（XD；aa 459-507）的野生型和 K496C 变体的深入分析，说明了这种方法的强大功能。使用自上而下的 MSMS 结合 TWIM，我们表明 XD 样品偶尔会表现出一种微观异质性，这种异质性无法从编码构建体的核苷酸序列中预测到，并且可能反映了在表达过程中发生的遗传漂移，无论是否为中性。此外，嫁接到 K496C XD 变体上的 1-氧代-2,2,5,5-四甲基-δ3-吡咯啉-3-甲基甲硫磺酸亚硝酮探针在电喷雾电离源中显示出氧化和/或质子化，导致人为的质量增加。
