Proteins play a vital role in carrying out various biological functions through interactions, such as the formation of protein complexes. The localization and microenvironments within cells are crucial for maintaining the structure and function of these complexes. Chemical cross-linking mass spectrometry (CXMS) is a technique used for analyzing protein complexes in living cells. However, it faces challenges such as complex spectra retrieval of cross-linked peptides and high cell disturbance.

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The Study

A team of researchers led by Prof. Zhang Lihua from Dalian Institute of Chemical Physics (DICP) has developed a new cross-linker for large-scale analysis of protein complexes in living cells. The study was published in Angewandte Chemie International Edition on March 30.

The researchers incorporated glycosidic bonds into the design of functional cross-linkers based on the high biocompatibility of glucose molecules and the mass spectrometry cleavable feature of glycosidic bonds. They screened and obtained trehalose, a highly biocompatible molecule, as the skeleton molecule and developed a mass spectrometry cleavable cross-linker, trehalose disuccinimidyl succinate (TDS).

This cross-linker showed superior cell viability maintenance compared to currently reported membrane-permeable chemical cross-linkers and enabled efficient cross-linking of protein complexes in cells under low disturbance conditions.


The researchers found that low-energy glycosidic bond–high-energy peptide bond mass spectrometry selective fragmentation mode reduced analysis complexity of the cross-linked peptide fragment spectra, significantly improving the efficiency and accuracy of cross-linked peptide identification. They identified conformation of 1,453 proteins corresponding to more than 3,500 cross-linked peptide pairs, and 843 protein-protein interaction information from Hela cells.

This new cross-linker enables in-vivo cross-linking of protein complexes in live cells, achieving large-scale and precise analysis. The glycosidic-bond-based mass-spectrometry-cleavable cross-linker improves the data analysis throughput and identification accuracy of cross-linking information with good amphiphilicity and biocompatibility.

The new cross-linker developed by the team of researchers led by Prof. Zhang Lihua from DICP has opened new avenues for large-scale analysis of protein complexes in living cells. The use of glycosidic bonds has improved the biocompatibility and mass spectrometry cleavable feature of cross-linkers. This technique will help in better understanding the conformation of proteins and their interactions with other proteins in living cells, leading to new discoveries in the field of biology.


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