Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers
Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers
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使用标签转移光交联剂快速绘制蛋白质相互作用图
DOI：
10.1002/ange.201809149
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发表时间：
2018
影响因子：
--
通讯作者：
Horne J
中科院分区：
文献类型：
--
作者：
Horne J
通过化学交联质谱法（XL-MS）分析蛋白质复合物受到可用交联剂的侧链反应性和大小、其缓慢的反应速率以及交联富集的困难的限制，特别是对于罕见的、瞬时的或动态的复合物。在这里，我们描述了两种新的XL试剂，其包含甲硫基磺酸盐（MTS）基团以标记引入诱饵蛋白的反应性半胱氨酸，以及基于残基无偏二氮丙啶的光活化XL基团以捕获其相互作用的配偶体。诱饵的还原去除将交联试剂的含巯基片段转移到目标上，该目标可以通过MS测序进行烷基化和定位并用于富集，从而能够检测低丰度的交联。使用这些试剂和定制的UV LED照射平台，我们表明在10秒内实现了最大交联产率。使用定义明确的肽/蛋白质调节相互作用（BID 80 - 102/MCL-1）和分子伴侣/底物复合物（Skp/OmpA）的动态相互作用界面证明了这种“标签和转移”方法的实用性。
Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue‐unbiased diazirine‐based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol‐containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well‐defined peptide/protein regulatory interaction (BID80‐102/MCL‐1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).