MOE Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200241, China.
J Mol Recognit. 2010 Jan-Feb;23(1):93-100. doi: 10.1002/jmr.995.
Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low-copy-number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre-fractionation were digested and then analyzed by LC-MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un-fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un-fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre-fractionation with MS-based proteomic analysis is simple, low-cost, and effective, providing the prospect of broad application in proteomics.
高效且高分辨率的蛋白质混合物分离，通常在胰蛋白酶消化和质谱（MS）分析之前进行，用于降低样品的复杂性，这种方法极大地提高了检测低拷贝数蛋白质的概率。我们实验室构建了一个由数千种具有不同蛋白质吸附效果的配体组成的亲和配体库。这些配体之间的结构差异导致不同的非键合的蛋白质-配体相互作用，因此每个配体对某些蛋白质组具有特定的亲和力。在这项工作中，我们首先选择了几种在蛋白质吸收谱中表现出较大谱带分布差异的合成亲和配体，然后将这些亲和配体串联起来，用于将复杂的大鼠肝胞质溶胶分成简单的亚群。最终，从串联亲和预分级收集的所有馏分都被消化，然后通过 LC-MS/MS 进行分析，这导致了 665 个独特的大鼠蛋白质组的高置信度鉴定，比未分级样品（371 个蛋白质组）检测到的蛋白质多 1.8 倍。其中，在串联馏分中鉴定出 375 个新蛋白质，而在未分级样品中鉴定出的蛋白质中有 290 个（80%）也出现在串联馏分中。最重要的是，430 个独特的蛋白质（64.7%）仅在特定的馏分中被鉴定，这表明串联亲和分级很好地分配了粗组织提取物。所有检测到的蛋白质都根据其理化特性（如 MW、pI、GRAVY 值、TM 螺旋）进行了生物信息学注释。这种方法突出了该方法对各种蛋白质类别的敏感性。串联亲和预分级与基于 MS 的蛋白质组学分析相结合，方法简单、成本低、效果好，为蛋白质组学的广泛应用提供了前景。
