Chen Chien-Sheng, Korobkova Ekaterina, Chen Hao, Zhu Jian, Jian Xing, Tao Sheng-Ce, He Chuan, Zhu Heng
Department of Pharmacology and Molecular Sciences & High Throughput Biology Center, Johns Hopkins University School of Medicine, Broadway Research Building 333, 733 Broadway, Baltimore, Maryland 21205, USA.
Nat Methods. 2008 Jan;5(1):69-74. doi: 10.1038/nmeth1148. Epub 2007 Dec 16.
Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.
尽管许多基因组已被解码，但仅针对芽殖酵母报道了包含单独纯化蛋白质的蛋白质组芯片，这主要是由于高通量蛋白质纯化的复杂性和难度。为了促进原核生物中的蛋白质组学研究，我们开发了一种高通量蛋白质纯化方案，该方案使我们能够在10小时内纯化大肠杆菌K12菌株编码的4256种蛋白质。然后将纯化的蛋白质点样到载玻片上以制备大肠杆菌蛋白质组芯片。我们使用这些芯片开发了用于鉴定参与识别DNA中潜在碱基损伤的蛋白质的检测方法。通过使用一组DNA探针，每个探针包含一个错配碱基对或一个无碱基位点，我们发现了少量能够以高亲和力和特异性识别每种类型探针的蛋白质。我们通过生化分析进一步评估了其中两种蛋白质，YbaZ和YbcN。包含具有特定修饰的DNA探针的文库的组装以及大肠杆菌蛋白质组芯片的可用性有可能揭示蛋白质与核酸之间重要的相互作用，而使用其他技术检测这些相互作用既耗时又困难。
