Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa–5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20–100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

化学交联反应 (XL) 是结构生物学中研究蛋白质-蛋白质相互作用 (PPI)（包括低丰度亚复合物）的重要策略。然而，选择 XL 试剂和条件很费力，并且主要仅限于分析可使用 SDS-PAGE 解析的蛋白质组装体。为了克服这些限制，我们在此开发了一种变性质量光度测定 (dMP) 方法，用于快速、可靠且用户友好的优化和监测化学 XL 反应。 dMP 是一种强大的两步实验方案，可确保在短短 5 分钟内实现 95% 的不可逆变性。我们证明，dMP 可在较宽的质量范围 (30 kDa–5 MDa) 内提供准确的质量鉴定，并对所有共存的 XL 物质（子复合物和聚集体）进行直接无标记相对定量。我们将 dMP 与 SDS-PAGE 进行比较，发现与基准不同，dMP 具有时间效率（3 分钟/三次），需要的材料显着减少（20–100×），并且提供单分子灵敏度。为了说明其在常规结构生物学应用中的实用性，我们证明 dMP 可以在 1 小时内筛选 20 个 XL 条件，准确识别和量化所有共存物种。综上所述，我们预计 dMP 将对更多 PPI 和大分子组装体、预期最终复合物以及途中形成的子复合物的结构表征能力产生影响。