Prof. TANG Shibing from Guangzhou Institutes of Biomedicine and Health (GIBH) of the Chinese Academy of Sciences, in collaboration with Prof. YANG Bing from Zhejiang University and Assoc. Prof. LIU Chao from Beihang University, developed a fluorosulfate containing enrichable and latent bioreactive unnatural amino acid to characterize protein-protein interactions in live cells. The study was published in Nature Communications. 

The identification of protein-protein interactions in live cell is important in the function study of proteins, and plays important roles in biomedical research. Proximity-enabled chemical cross-linkable unnatural amino acids (Uaas) have been used for the in situ identification of protein interactions in live cells via their ability to capture weak and transient protein interactions. 

Identification of cross-linked peptides with mass spectrometry is critical to ensure the specificity of interacting protein identification and to reveal the protein interaction interfaces. However, due to the complexity of protein samples and data analysis process, it is challenging to perform high-throughput identification of Uaa-mediated cross-linked peptides in live cells. Enrichment of cross-linked peptides before mass spectrometry analysis can improve the identification efficiency.

In this study, researchers developed an fluorosulfate containing latent bioreactive unnatural amino acid eFSY, shorted for enrichable FluoroSulfate-L-tyrosine. The eFSY can be incorporated into specific site of target proteins via genetic code expansion. The fluorosulfate of eFSY covalently cross-linked with tyrosine, histidine, or lysine in interaction proteins via a proximity-enabled sulfur-fluoride exchange (SuFEx) reaction. The alkyne group of eFSY can be biotinylated by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and be used to enrich cross-linked peptides at the peptide level, thus improving the sensitivity of cross-linked peptides identification. 

In addition, researchers demonstrated that eFSY mediated cross-linking is fragmented in different patterns in mass spectrometry. While the sulfamate bonds formed by cross-linking with histidine and lysine are mass spectrometry cleavable, the cross-linking bond with tyrosine is not. 

To utilize this characteristic of eFSY cross-linking product, the cross-linking identification software AixUaa was developed. AixUaa is compatible with both cleavable and non-cleavable modes at the same time, which can precisely match the cross-linking peptides and sites, and increase the number of identified cross-linked peptides. Researchers systematically identified direct interactomes of Thioredoxin 1 (Trx1) and Selenoprotein M (SELM) with eFSY and AixUaa in Escherichia coli and mammalian cells separately.

This study provides a promising method for protein-protein interaction study.