A strategy for rapid, high-confidence protein identification.
A strategy for rapid, high-confidence protein identification.
复制标题
快速、高可信度蛋白质鉴定的策略。
DOI：
10.1021/ac970488v
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发表时间：
1997
影响因子：
7.4
通讯作者：
Chait,BT
中科院分区：
文献类型：
--
作者：
Qin,J;Fenyö,D;Zhao,Y;Hall,WW;Chao,DM;Wilson,CJ;Young,RA;Chait,BT
描述了一种使用基质辅助激光解吸/电离串联离子陷阱质谱仪与基因组数据库搜索策略相结合的快速、高置信度鉴定蛋白质的方法。该程序包括从凝胶中切除铜染色带或斑点，凝胶内胰酶消化蛋白质，对生成的胰蛋白酶多肽混合物进行单级质谱分析，然后对选定的单个多肽进行串联离子陷阱质谱分析，以及使用PepFrag程序搜索相关基因组数据库。该方案提供了灵敏的、实时的蛋白质识别以及简单的修饰识别。一个操作员每天可以毫不含糊地从一个生物体中识别出5个−10蛋白质，该生物体的基因组在凝胶上装载的蛋白质水平为>；0.5pmol。通过对人HTLV-I制剂的一条带和酵母RNA聚合酶II C末端重复结构域亲和制剂的11种不同蛋白质的鉴定和鉴定，证明了该技术的实用性。这项技术在后基因组生物科学中具有巨大的潜力，有望促进大分子组合的解剖、疾病状态标记的定义以及生物过程中蛋白质靶标的研究，如细胞周期和信号转导。
A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5−10 proteins/day from an organism whose genome is known at a level of >0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.