A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

• Published in: Molecular & cellular proteomics Vol. 13; no. 12; pp. 3533 - 3543
• Main Authors: Kaake, Robyn M., Wang, Xiaorong, Burke, Anthony, Yu, Clinton, Kandur, Wynne, Yang, Yingying, Novtisky, Eric J., Second, Tonya, Duan, Jicheng, Kao, Athit, Guan, Shenheng, Vellucci, Danielle, Rychnovsky, Scott D., Huang, Lan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2014; The American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Biotin - chemistry; Cattle; Cross-Linking Reagents - chemical synthesis; Cytochromes c - metabolism; HEK293 Cells; Humans; Molecular Sequence Data; Protein Binding; Protein Interaction Mapping - instrumentation; Protein Interaction Mapping - methods; Proteome - metabolism; Staining and Labeling - methods; Tandem Mass Spectrometry - instrumentation; Tandem Mass Spectrometry - methods
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.M114.042630

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.