Table4_Enhanced protein–protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.XLSX

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein–protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.

化学交联结合质谱法（chemical cross-linking coupled with mass spectrometry）已成为一种高效研究策略，可在复杂生物系统中实现对带有直接相互作用界面的蛋白质相互作用组的全谱分析。炔基标记可富集交联剂（alkyne-tagged enrichable cross-linkers）可提升低丰度交联肽段的覆盖度，因而常结合点击化学（click chemistry）进行生物素偶联，以实现交联肽段的富集。然而，目前仍缺乏针对点击化学富集策略（基于蛋白质层面或肽段层面）以及多种可裂解点击化学配体（cleavable click-chemistry ligands，包括酸解、还原、光解类型）在交联肽段富集与释放过程中效率的系统性评价。本研究中，我们结合炔基标记交联剂的体内化学交联手段，分别在蛋白质层面与肽段层面探索了搭载三种可裂解点击化学配体的基于点击化学的富集策略。通过对比分析，我们证实搭载酸裂解标签（acid-cleavable tags）的基于蛋白质层面的点击化学偶联策略可实现最多的交联肽段鉴定数量。该策略的优化升级推动了全蛋白质组交联分析的发展，最终在1871种细胞内丰度分布广泛的蛋白质之间构建了包含5518个蛋白质-蛋白质相互作用对的网络（protein–protein-interaction network）。综上，本研究结果为高效实现交联肽段富集提供了指导性方案，有助于推动蛋白质相互作用组的深度表征以开展功能分析研究。