Cross-linking mass spectrometry: Workflow enhancements for mapping large-scale interactomes

In vivo chemical cross-linking combined with mass spectrometry analysis (XL-MS) enables the direct capture of protein-protein interactions (PPIs) within their native cellular environment. This platform provides a unique capability to capture weak or transient interactions in near-native contexts, complementing other large-scale methods that may lose these associations during cell lysis or purification. The workflow involves selection of suitable chemical cross-linkers to stabilize PPIs in biological samples, ranging from organelles and cells to whole tissues. This is followed by careful sample preparation, high-resolution MS analysis, and computational identification of cross-linked peptides. Continuous advancements at each stage of this workflow have enhanced the sensitivity, throughput, and accuracy of the technique. A pivotal development has been the design of novel multifunctional cross-linkers featuring improved cell permeability, MS-cleavable sites, enrichment tags, and multisite reactivity. Furthermore, recent advances in MS instrumentation, data acquisition strategies, and computational software have increased the depth and reliability of cross-link identification. This review focuses on general workflows and recent innovations that accelerate the impact of in vivo XL-MS, highlighting experimental strategies for proteome-wide PPI mapping. The successful standardization of methodology would enable widespread acceptance and application of XL-MS to address diverse and complex biological questions.