Neubauer G, Gottschalk A, Fabrizio P, Séraphin B, Lührmann R, Mann M
European Molecular Biology Laboratory, Heidelberg, Germany.
Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):385-90. doi: 10.1073/pnas.94.2.385.
Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B' homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.
在此，我们通过在基因组序列数据库中搜索质谱数据，报告了从酿酒酵母中快速鉴定剪接体U1小核核糖核蛋白（snRNP）的蛋白质。含有组氨酸标签的70K蛋白的U1 snRNP通过抗m3G帽免疫亲和及随后的镍亚氨基三乙酸色谱法从细胞提取物中分离出来。获得了一个含有20种蛋白质的U1 snRNP组分。通过甘油梯度离心进一步纯化鉴定出9种U1 snRNP特异性蛋白和6种常见蛋白。通过纳升电喷雾质谱对U1 snRNP蛋白进行了部分测序，并通过多个肽序列标签在数据库中鉴定了它们的基因。除了已知的常见蛋白D1、D3、F和G外，还鉴定出了D2和E的同源物。在单独纯化和分析的核心U2 snRNP中检测到了相同的6种常见蛋白。这6种蛋白与酵母snRNPs的生化关联在此首次展示。有趣的是，未检测到Sm B/B'同源物。除了已充分表征的酵母U1特异性蛋白[U1 - 70K（Snp1p）、U1 - A（Mud1p）、Prp39p和Prp40p]外，还鉴定出了U1 - C蛋白的同源物以及另外4种新的U1特异性蛋白，这些蛋白在哺乳动物U1中未发现。这是首次通过质谱对来自具有测序基因组的生物体的多蛋白复合物的成分进行表征。该技术应适用于任何可从已知基因组的生物体中进行生化纯化的蛋白复合物。
