Capillary zone electrophoresis for analysis of complex proteomes using an electrokinetically pumped sheath flow nanospray interface.
Capillary zone electrophoresis for analysis of complex proteomes using an electrokinetically pumped sheath flow nanospray interface.
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毛细管区电泳，用于使用电动泵送的护套纳米喷雾界面分析复杂蛋白质组织。
DOI：
10.1002/pmic.201300295
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发表时间：
2014-03
期刊：
影响因子：
3.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Sun, Liangliang;Zhu, Guijie;Yan, Xiaojing;Champion, Mathew M.;Dovichi, Norman J.
绝大多数蛋白质组学研究采用反相高效液相色谱与串联质谱联用来分析细胞裂解物的胰蛋白酶消化物。这项技术相当成熟，通常可以识别数百到数千种肽，从而推断数百到数千种蛋白质的身份。这些方法通常需要毫克到微克的起始材料。毛细管区带电泳提供了一种有趣的替代分离方法，其分离机制与 HPLC 不同。 25 年前，毛细管电泳在蛋白质分析中受到了一些关注。由于电喷雾接口的性能有限以及那个时代质谱仪的速度和灵敏度有限，这些努力陷入停滞。本综述考虑了一种与 Orbitrap Velos 和线性 Q-trap 质谱仪相结合的新电喷雾接口设计。毛细管区带电泳与该接口和这些检测器相结合，可以在不到一小时的时间内对大肠杆菌消化物中的 > 1,250 种肽进行单次检测，在六小时的总分析时间内从大肠杆菌消化物固相萃取产生的七个级分的分析中鉴定出近 5,000 种肽，对标准蛋白质生成的肽进行低阿托摩尔检测限，并对肽的选定离子监测进行高 zeptomole 检测限。集成的在线固定胰蛋白酶微反应器可以消化和分析皮克量的复杂真核蛋白质组。
The vast majority of proteomic studies employ reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry for analysis of the tryptic digest of a cellular lysate. This technology is quite mature, and typically provides identification of hundreds to thousands of peptides, which is used to infer the identity of hundreds to thousands of proteins. These approaches usually require milligrams to micrograms of starting material. Capillary zone electrophoresis provides an interesting alternative separation method based on a different separation mechanism than HPLC. Capillary electrophoresis received some attention for protein analysis beginning 25 years ago. Those efforts stalled because of the limited performance of the electrospray interfaces and the limited speed and sensitivity of mass spectrometers of that era. This review considers a new electrospray interface design coupled with Orbitrap Velos and linear Q-trap mass spectrometers. Capillary zone electrophoresis coupled with this interface and these detectors provides single shot detection of >1,250 peptides from an E. coli digest in less than one hour, identification of nearly 5,000 peptides from analysis of seven fractions produced by solid-phase extraction of the E. coli digest in a six hour total analysis time, low attomole detection limits for peptides generated from standard proteins, and high zeptomole detection limits for selected ion monitoring of peptides. Incorporation of an integrated on-line immobilized trypsin microreactor allows digestion and analysis of picogram amounts of a complex eukaryotic proteome.
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影响因子：
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