UC Irvine

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 new sample preparation strategies and high-resolution mass spectrometry. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform allows us to derive proteome wide protein interaction networks both in vitro and in vivo. We further expanded the use of this platform to obtain PPIs.

Many complicated organ related diseases such as heart failure or obesity are not easily replicated in cell culture models. Additionally, a lack of information on protein complexes within these tissues limits our understanding of disease pathways and ways to treat it. Few studies have been able to perform large-scale XL-MS from tissue, due in part to the overwhelming complexity of tissue samples. We have expanded our XL-MS platform to derive in vivo protein interaction networks from mouse brain, heart, liver, lung, and kidney.

Photochemistry has shown great potential in recent years to capture interaction sites not targetable by specific linkers. The alkyl diazirine is particularly attractive due to small size, photostability, and success in XL-Ms studies. Due to the complexity of the searches, identifying cross-link products can still be challenging. We developed an MS-cleavable heterobifunctional photoreactive and enrichable cross-linker to facilitate non-specific XL-MS.