Chen Jinzhi, Balgley Brian M, DeVoe Donald L, Lee Cheng S
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Anal Chem. 2003 Jul 1;75(13):3145-52. doi: 10.1021/ac034014+.
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 approximately 240. 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 microg. 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 microm to 100 microm, the required protein loading is further decreased from 9.6 microg 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.
开发了一种集成蛋白质组浓缩/分离方法，该方法涉及毛细管等电聚焦（CIEF）与毛细管反相液相色谱（CRPLC）的在线联用，可对蛋白质和肽混合物提供显著的分析物浓缩以及极高的分离能力。在分析物聚焦完成后，自锐化效应极大地限制了分析物扩散，并有助于将分析物堆积在聚焦狭窄的条带中，浓缩因子约为240。除了分析物聚焦外，CIEF作为第一分离维度，根据蛋白质/肽的pI差异进行分离，并且比强阳离子交换色谱具有更高的分离能力。CIEF和CRPLC这两种高分辨率且完全正交的分离技术相结合，再加上分析物聚焦和浓缩，显著提高了传统质谱对低丰度蛋白质鉴定的动态范围和灵敏度。基于CIEF的多维分离/浓缩平台能够鉴定出比文献中报道的方法更多的酵母可溶性蛋白质，而仅需9.6微克的蛋白质上样量。该蛋白质上样量比已报道的非凝胶基蛋白质组技术所采用的上样量低2 - 3个数量级。所鉴定酵母蛋白质的密码子适应指数值分布接近整个酵母蛋白质组的预测值，支持基于CIEF的蛋白质组分离技术实现全面蛋白质组分析的能力。通过将色谱柱内径从180微米减小到100微米，所需的蛋白质上样量进一步从9.6微克降至960纳克，这说明了这种蛋白质组技术在分析小细胞群体或有限组织样本中的蛋白质谱方面的潜在用途。
