Shotgun cross-linking analysis for studying quaternary and tertiary protein structures
Shotgun cross-linking analysis for studying quaternary and tertiary protein structures
复制标题
DOI：
10.1021/pr070234i
复制
发表时间：
2007-10-01
影响因子：
4.4
通讯作者：
Phinney, Brett S.
中科院分区：
文献类型：
--
作者：
Lee, Young Jin;Lackner, Laura L.;Phinney, Brett S.
我们开发了一种新的方法，该方法采用了一种新的计算机算法，用于化学交联蛋白或多蛋白复合物的三级和四级相互作用位点的敏感和高通量分析。首先，我们仅使用高精度LC-MS/MS数据直接分析化学交联蛋白的消化产物。我们用计算机算法分析这些数据，我们称之为X！链接，查找两个肽之间的交联。我们的算法快速，只需几秒钟就可以分析类似于5000 MS/MS的光谱。我们应用该算法分析了细胞色素c和线粒体分裂动力蛋白突变体Dnm1G385D中使用氨基特异性试剂BS3化学生成的交联位点，Dnm1G385D作为稳定的同型二聚体存在。从细胞色素c（一种成熟的测试蛋白）中，我们从单个LC-MS/MS数据集的257个MS/MS光谱中共鉴定出31个交联，21个肽间交联和10个肽内交联。该技术的高灵敏度表明，细胞色素c中的所有19个赖氨酸都被检测为交联产物，并且在20a内的所有赖氨酸对中有33%也被观察到为交联产物。对Dnm1G385D的二聚体进行交联分析，在单个LC-MS/MS数据集的98个MS/MS光谱中鉴定出46个交联，38个肽间交联和8个肽内交联。这些结果代表了迄今为止在单个蛋白质或蛋白质二聚体中鉴定的最丰富的交联。统计分析表明，优化过滤参数后，虚假发现率为1%。对使用我们的方法识别的交联的进一步分析表明，当存在多个可交联位点或几个相似序列时，仔细的人工检查对于正确分配交联位点是重要的。总之，我们开发了一种敏感的基于质谱的方法来鉴定肽-肽交联，不需要同位素标记或与非交联对照进行比较，使其比目前的方法更快、更简单。
We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze similar to 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide crosslinks, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide crosslinks, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.