Department of Chemistry, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy.
J Chromatogr A. 2011 Dec 9;1218(49):8760-76. doi: 10.1016/j.chroma.2011.05.094. Epub 2011 Jun 6.
Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.
质谱联用与各种分离方法相结合是蛋白质组学的主要方法。在自上而下的方法中，蛋白质用特定的蛋白酶切割，然后进行肽分离和 MS 鉴定。在自下而上的方法中，完整的蛋白质被引入质谱仪。然后，通过电喷雾电离产生的离子经受气相分离、碎裂、片段分离和质谱和色谱数据的自动解释，从而获得完整蛋白质的分子量和蛋白质片段模式。这种方法需要能够分离蛋白质的多电荷同位素簇的高精度质量测量分析仪，例如混合离子阱-傅里叶变换仪器 (LTQ-FTICR、LTQ-Orbitrap)。针对蛋白质定制的前端分离技术对于实现自上而下的蛋白质组学至关重要。本文旨在提供适用于 MS 偶联的蛋白质色谱和电泳分离方法的最新技术，说明用于 LC-MS 自上而下蛋白质组学的单维方法和多维方法。此外，还讨论了蛋白质色谱学的一些最新进展，这些进展可能为目前使用的方法提供替代方案。
