Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography.
Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography.
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DOI：
10.1074/mcp.m111.014126
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发表时间：
2012-03
期刊：
影响因子：
--
通讯作者：
Aebersold R
中科院分区：
文献类型：
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作者：
Leitner A;Reischl R;Walzthoeni T;Herzog F;Bohn S;Förster F;Aebersold R
化学交联结合质谱分析提供了从蛋白质和蛋白质复合物获得低分辨率结构信息的可能性。通过交联连接的肽的鉴定为氨基酸侧链的物理相互作用提供了直接证据，这些信息可用于计算建模目的。尽管近年来取得了令人印象深刻的进展，但实验观察到的交联的数量仍然低于交联剂跨度内交联剂侧链的可能接触的数量福尔斯。在这里，我们提出了两个互补的实验策略，以扩大交联数据集。首先，通过尺寸排阻色谱法富集交联肽基于其较高分子量选择交联肽，从而耗尽交联样品的蛋白水解酶中存在的大部分未修饰肽。其次，我们证明，除了胰蛋白酶，如Asp-N，蛋白酶的使用，可以额外增加可观察到的交联位点的数量。SEC富集和多蛋白酶酶的好处是证明了一组模型蛋白质和改进的工作流程适用于表征的20 S蛋白酶体从兔和裂殖酵母粟酒裂殖酵母。
Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.