Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels
Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels
复制标题
DOI：
10.1073/pnas.93.25.14440
复制
发表时间：
1996-12-10
影响因子：
11.1
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Shevchenko, A;Jensen, ON;Mann, M
通过基因组测序发现的许多未表征的开放阅读框的功能可以在表达的基因产物蛋白质组水平上确定。然而，从微量蛋白质中识别同源基因一直是分子生物学的主要问题之一。以酵母为例，我们在这里证明，质谱蛋白质鉴定是一个完整的测序基因组的这个问题的一般解决方案。作为第一个屏幕，我们的策略使用自动激光解吸电离质谱的肽混合物产生的蛋白质的凝胶内胰蛋白酶消化。高达90%的蛋白质是通过搜索序列数据库的肽质量列表获得高精度。通过纳米电喷雾串联质谱法对未分离的混合物的几种肽进行部分测序，然后用多个肽序列标签进行数据库搜索来鉴定剩余的蛋白质。在盲法试验中，该方法在所有情况下都能进行明确的鉴定。在迄今为止最大的单个蛋白质鉴定项目中，共成功分析了150个凝胶点，其中许多在亚皮摩尔范围内，大大扩大了酵母二维凝胶数据库。超过32种蛋白质是新的，并与酵母基因组中以前未表征的开放阅读框架相匹配。本研究确定，质谱法提供了所需的吞吐量，确定性的鉴定，并作为选择的方法，连接基因组和蛋白质组的一般适用性。
The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags, In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots-many of them at subpicomole amounts-were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.