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首页> 外文期刊>Journal of proteome research >Shotgun cross-linking analysis for studying quaternary and tertiary protein structures
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Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

机译：gun弹枪交联分析，用于研究四级和三级蛋白质结构

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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.
机译：我们开发了一种新方法，该方法采用新颖的计算机算法对来自化学交联蛋白或多蛋白复合物的第三级和第四级相互作用位点进行灵敏且高通量的分析。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物。我们使用称为X！Link的计算机算法分析这些数据，以找到两个肽之间的交叉链接。我们的算法快速，只需几秒钟即可分析类似于5000 MS / MS的光谱。我们应用此算法来分析使用氨基特异性试剂BS3在细胞色素c和线粒体分裂动力蛋白突变体Dnm1G385D中化学生成的交联位点，该突变体以稳定的同型二聚体形式存在。从细胞色素c（一种完善的测试蛋白）中，我们从一个LC-MS / MS数据集中的257个MS / MS谱图中鉴定出总共31个交联键，21个肽间键合和10个内肽交联键。该技术的高灵敏度由以下事实表明：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20 A内的所有Lys对中也有33％被观察为交联。在单个LC-MS / MS数据集中的98 MS / MS光谱中，对Dnm1G385D的交联二聚体形式的分析确定了总共46个交联，38个肽间交联和8个内肽交联。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出的最丰富的交联。统计分析表明，优化过滤参数后，错误发现率为1％。使用我们的方法对所确定的交联点的进一步分析表明，当存在多个可交联位点或几个类似序列时，仔细的手动检查对于正确分配交联点很重要。总而言之，我们开发了一种基于质谱的灵敏方法，可识别不需要同位素标记或无需与非交联对照进行比较的肽-肽交联，从而使其比现有方法更快，更简单。

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