期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2023360118
关键词
in vivo cross-linking mass spectrometry; protein-protein interactions; Alkyne-A-DSBSO; click chemistry enrichment; proteome-wide XL-MS
资金
- NIH [R01GM074830, R01GM130144]
A robust in vivo XL-MS platform has been developed to map protein-protein interactions at a systems level, enabling the construction of the largest in vivo PPI network to date from HEK 293 cells. This advanced strategy provides a highly detailed panoramic portrait of human interactomes associated with diverse cellular pathways and can be generalized for charting protein interaction landscapes in any organisms.
Defining protein-protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking-mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine-lysine linkages from in vivo crosslinked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.
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