Top-Down Proteomics by Means of Orbitrap Mass Spectrometry
Top-Down Proteomics by Means of Orbitrap Mass Spectrometry
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DOI：
10.1007/978-1-4939-0685-7_31
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发表时间：
2014-01-01
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影响因子：
--
通讯作者：
Scheffler, Kai
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文献类型：
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作者：
Scheffler, Kai
自上而下的蛋白质组学已经成为分析完整蛋白质的流行方法。术语“自上而下”已被创造用于不涉及任何酶或化学裂解的蛋白质分析，而是将蛋白质电离为健全分子，并对质谱仪内解离后产生的完整物质及其碎片离子进行质量分析。蛋白质的一个或几个电荷状态被质量分离并在气相中进行解离（MS/MS）。获得的片段质量，主要来自蛋白质沿着其氨基酸骨架的裂解，与完整蛋白质直接相关。使用生物信息学工具，根据蛋白质的大小和获得的片段离子的数量，将片段质量与已知的蛋白质序列相匹配，或者可以用于部分或完全从头测序。此外，这种方法提供了关于蛋白质修饰状态的全局信息，包括同种型的数量和类型及其化学计量，并允许在氨基酸序列内精确定位修饰。单一纯化蛋白质的自上而下分析可通过基质辅助激光解吸电离或电喷雾电离在直接输注时进行，而无需在线色谱分离，而复杂蛋白质混合物的自上而下分析使得预分级分离与在线耦合到质谱仪的有效前端色谱分离相结合是不可避免的。
Top-down proteomics has become a popular approach for the analysis of intact proteins. The term "top down" has been coined for the analysis of proteins not involving any enzymatic or chemical cleavage but rather the ionization of the protein as a sound molecule and mass analysis of intact species and fragment ions thereof produced upon dissociation inside a mass spectrometer. One or several charge states of the protein are mass-isolated and subjected to dissociation (MS/MS) in the gas phase. The obtained fragment masses, predominantly from cleavages of the protein along its amino acid backbone, are directly related to the intact protein. Using bioinformatics tools the fragment masses are matched against a known protein sequence or can alternatively be used for partial or full de novo sequencing, depending on the size of the protein and the number of fragment ions obtained. Moreover, this approach provides global information about modification states of proteins including the number and types of isoforms and their stoichiometry and allows for the precise localization of modifications within the amino acid sequence. Top-down analysis of a single, purified protein can be performed by matrix-assisted laser desorption ionization or electrospray ionization upon direct infusion without online chromatographic separation, whereas top-down analysis of complex protein mixtures makes pre-fractionation combined with an efficient front-end chromatographic separation coupled online to the mass spectrometer inevitable.