Miniaturized formats for efficient mass spectrometry-based proteomics and therapeutic development.
Miniaturized formats for efficient mass spectrometry-based proteomics and therapeutic development.
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用于高效基于质谱的蛋白质组学和治疗开发的小型化形式。
DOI：
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发表时间：
2002
影响因子：
3.4
通讯作者：
N. Kelleher
中科院分区：
文献类型：
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作者：
G. Valaskovic;N. Kelleher
用于电喷雾电离质谱（ESI-MS）的离线小型化“纳米喷雾”格式使得能够常规鉴定飞摩尔量的蛋白质或肽。使用在线小型化ESI-MS方法（例如纳米孔LC-MS和CE-MS）已经实现了更大的进步。在线方法能够实现更高的灵敏度（亚阿托摩尔检测限）、动态范围和通量。在用于蛋白质分析的离线或在线方法中，通常以“自下而上”的方法分离样品并酶促消化，用肽片段的MS分析，产生5-50%的序列覆盖率。获得生物学相关的（结构/功能）信息（如错误区域或跨国后修饰的定位）通常需要100%的序列覆盖率，这可以通过MS分析完整的蛋白质与“自上而下”的方法来获得。自上而下的方法在蛋白质组学中的广泛成功将需要开发新的小型化样品制备方法，沿着新的生物信息学工具。随着这些小型化格式继续为蛋白质组学应用提供动力，它们无疑将为LC-MS中从药物发现到开发的“交叉”应用提供支持。使用比通常所需少一个数量级的样品进行代谢物鉴定的示例，同时信号增加一个数量级，说明了小型化格式在铅表征活动中的潜力。
Off-line miniaturized "nano-spray" formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50% sequence coverage, in a "bottom-up" approach. Obtaining biologically relevant (structure/function) information (such as the localization of regions of error or post-transnational modifications) often demands 100% sequence coverage and this may be obtained by analyzing intact proteins by MS with a "top-down" methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate "cross-over" applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.