Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry
Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry
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DOI：
10.1021/ac990986z
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发表时间：
2000-02-01
影响因子：
7.4
通讯作者：
Thibault, P
中科院分区：
文献类型：
--
作者：
Li, JJ;Kelly, JF;Thibault, P
耦合的微加工设备的纳米电喷雾质谱仪使用三重四极杆和四极杆飞行时间质谱仪（QqTOF MS）的痕量级膜蛋白的分析。短的一次性纳米电喷雾发射器通过低死体积连接直接耦合到芯片装置。该集成装置的分析性能-在灵敏度和重现性方面进行了评价标准肽混合物。在选择的离子监测中实现了不同肽的浓度检测限范围从3.2到43.5 nM，因此与使用相同芯片/质谱仪的微电喷雾相比，灵敏度提高了10倍。重复进样表明，迁移时间的重现性通常小于3.1% RSD，而峰面积的RSD值为6-13%。虽然对于复杂的质谱通常不能实现单个组分的完全解析，但是本发明的芯片毛细管电泳（chip-CE）装置能够在质谱鉴定之前进行适当的样品净化和多组分样品的部分分离。蛋白质电泳分析通常在1.5分钟内完成，峰宽为1.8-2.5秒（半高定义），如单个重建离子电泳图所示。该芯片CE/QqTOF MS系统的应用程序进一步证明了流感嗜血杆菌蛋白质组的一个子集，形成的膜蛋白的鉴定。首先通过一维凝胶电泳分离的条带被切除并消化，提取的胰蛋白酶肽被加载到芯片上，无需任何进一步的样品净化或在线吸附预浓缩。精确的分子量测定（
The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly,coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device-in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (