Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis
Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis
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DOI：
10.1021/ac034014
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发表时间：
2003-07-01
影响因子：
7.4
通讯作者：
Lee, CS
中科院分区：
文献类型：
--
作者：
Chen, JZ;Balgley, BM;Lee, CS
开发了一种包括毛细管等电聚焦（CIEF）和毛细管反相液相色谱（CRPLC）在线组合的集成蛋白质组浓缩/分离方法，用于提供显著的分析物浓缩和对蛋白质和肽混合物的极高分辨能力。分析物聚焦完成后，自锐效应极大地限制了分析物扩散，并有助于分析物堆积在浓度因子接近240的窄聚焦带中。除分析物聚焦外，CIEF作为第一分离维度，可根据蛋白质/肽的pI差异进行分离，并提供比强阳离子交换色谱更高的分离能力。CIEF和CRPLC的两种高分辨和完全正交的分离技术的分组，连同分析物聚焦和浓缩，显著提高了传统质谱法对低丰度蛋白质的鉴定的动态范围和灵敏度。基于CIEF的多维分离/浓缩平台能够识别比文献中提出的方法更多的酵母可溶性蛋白，但仅需要9.6 μ g的蛋白质加载。这种蛋白质加载比所报道的非基于凝胶的蛋白质组技术所采用的蛋白质加载低2-3个数量级。所鉴定的酵母蛋白质的密码子适应指数值的分布接近于整个酵母蛋白质组的预测值，并且支持基于CIEF的蛋白质组分离技术用于实现全面蛋白质组分析的能力。通过将色谱柱的内径从180 μ m减小到100 μ m，所需的蛋白质加载量从9.6 μ g进一步减小到960 ng，说明了这种蛋白质组技术用于分析小细胞群体或有限组织样品中的蛋白质谱的潜在用途。
An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of similar to240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/ peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 mug. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 mum to 100 mum, the required protein loading is further decreased from 9.6 mug to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.