Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes
Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes
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DOI：
10.1074/mcp.t400016-mcp200
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发表时间：
2005-08-01
影响因子：
7
通讯作者：
Borchers, CH
中科院分区：
文献类型：
--
作者：
Petrotchenko, EV;Olkhovik, VK;Borchers, CH
研究复合物中蛋白质-蛋白质相互作用的一种新兴方法是结合化学交联和对复合物蛋白水解后获得的交联肽（交联）进行质谱分析。然而，这种方法具有若干挑战和限制，包括难以检测交联、来自非信息性“交联肽”（死端和肽内交联）的潜在干扰以及通过质谱法明确鉴定交联。因此，我们已经合成了同位素编码的乙二醇双（琥珀酰亚胺基琥珀酸酯）衍生物（D-12-EGS），其含有12个氘原子，当与其H-12对应物以1：1的混合物施用时易于检测交联，并且还可裂解以释放交联肽，从而允许通过MS测序进行明确鉴定。此外，水解裂解允许快速区分不同类型的交联。死端交联的裂解产生双峰，峰间距为4.03 Da，较低的峰出现在比原始交联肽的H-12形式的质量低162 Da的分子量处。肽内交联的裂解导致双峰相距8.05 Da，并且比原始交联肽的H-12形式的分子量低62 Da。肽间交联的裂解形成一对4.03-Da的双峰，由于交联剂的连接部分，每对的较低质量成员各自从其未修饰的分子量上移82 Da。所有这些信息已被纳入一个软件算法，允许自动筛选和检测基质辅助激光解吸/电离质谱中的交联和交联类型。总之，通过使用同位素编码的可切割的交联剂和我们的软件算法，随后通过裂解后的交联肽的质谱测序，这些物质的检测的容易性已被证明是用于研究多组分蛋白质复合物的有力工具。
An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the crosslinked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" ( dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis( succinimidylsuccinate) derivate (D-12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1: 1 mixture with its H-12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H-12 form of the original crosslinked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H-12 form of the original crosslinked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.