Structural Analysis of a Prokaryotic Ribosome Using a Novel Amidinating Cross-Linker and Mass Spectrometry

The structure of the Escherichia coli ribosome, a 2.5 MDa ribonucleoprotein complex containing more than 50 proteins, was probed using the novel amidinating cross-linker diethyl suberthioimidate (DEST) and mass spectrometry. Peptide cross-links derived from this complex structure were identified at high confidence (FDR 0.8%) from precursor mass measurements and collision-induced dissociation (CID) fragmentation spectra. The acquired cross-linking data were found to be in excellent agreement with the crystal structure of the E. coli ribosome. DEST cross-links are particularly amenable to strong cation exchange (SCX) chromatography, facilitating a large-scale analysis. SCX enrichment and fractionation were shown to increase the number of cross-link spectra matches in our analysis 10-fold. Evidence is presented that these techniques can be used to study complex interactomes.

本研究以分子量为2.5 MDa的大肠杆菌（Escherichia coli）核糖体为研究对象，该核糖体是一类包含50余种蛋白质的核糖核蛋白复合物。本研究采用新型氨基化交联剂二乙基辛二酰二硫代亚氨酸酯（diethyl suberthioimidate, DEST）结合质谱法对其结构进行解析。通过前体质量测量与碰撞诱导解离（collision-induced dissociation, CID）碎裂谱图分析，我们以0.8%的错误发现率（FDR）实现了该复合物结构衍生肽交联产物的高置信度鉴定。所获得的交联质谱数据与大肠杆菌核糖体的晶体结构具有高度一致性。DEST交联产物尤其适配强阳离子交换（strong cation exchange, SCX）色谱法，可有效支撑大规模交联分析。实验证实，强阳离子交换富集与分级分离步骤可将本研究中的交联谱图匹配数量提升10倍。本研究证明，此类技术可用于解析复杂的蛋白质相互作用组。