Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics
Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics
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DOI：
10.1021/ac0011336
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发表时间：
2001-04-15
影响因子：
7.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Shen, YF;Zhao, R;Smith, RD
在这项研究中，高效填充毛细管反相液相色谱（RPLC）耦合在线与高性能傅立叶变换离子回旋共振（FTICR）质谱已被研究的复杂的细胞蛋白水解酶的表征。填充有小（3 μ m）C18键合颗粒的长毛细管柱（80-cm）在10 000 psi的压力下在组成梯度条件下与ESI-FTICR质谱仪连接时提供了细胞蛋白水解多肽的类似于1000的总峰容量。（例如，500 μ g）可以装载到内径为150 μ m的填充毛细管上，而不会显著损失分离效率。发现具有合适内径的预柱对于改善洗脱再现性而不显著损失分离质量是有用的。多孔颗粒填充的毛细管被发现提供更好的结果比那些含有无孔颗粒，因为它们的更高的样品容量。来自填充毛细管RPLC与高分辨率FTICR的组合的二维分析产生用于分离>1百万个多肽组分的组合能力，并且同时提供用于基于先前描述的准确质量标签概念鉴定分离组分的信息。
In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-mum) C18 bonded particles provided a total peak capacity of similar to 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10 000 psi, Large quantities of cellular proteolytic digests (e.g,, 500 mug) could be loaded onto packed capillaries of 150-mum inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of >1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.