Proteins carry out specific biological functions in cells through conformational changes and interactions. Therefore, precise, in situ analysis of protein complex changes is essential for understanding cellular functions, uncovering disease mechanisms, and identifying potential drug targets.
Recently, a research team led by Prof. ZHANG Lihua from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS) developed a novel dimethylpiperidine-based cross-linker that enables one-step enrichment and quantitative analysis of protein complexes. This cross-linker minimizes sample loss and enables in vivo cross-linking mass spectrometry (XL-MS) analysis using as few as 10,000 cells.
This study was published in Angewandte Chemie International Edition.
In vivo XL-MS has emerged as a powerful technique in recent years for studying protein complexes in living cells. However, most current methods suffer from considerable sample loss during the enrichment of cross-linked peptides, due to complicated, multiple steps. This limits their application in analyzing the limited sample and hampers reproducibility in quantitative analysis.
To overcome this limitation, the researchers designed a novel cross-linker, 2,6-dimethylpiperidine disuccinimidyl tridecanoate (DPST), using dimethylpiperidine as the enrichment handle. DPST exhibits cell membrane permeability, enrichment capability, and quantitative functionality.
By leveraging TMT antibodies, DPST enables one-step enrichment and reversible elution of cross-linked peptides, eliminating sample loss from traditional multi-step processes. This allows reliable XL-MS analysis of as few as 10,000 cells. Moreover, the DPST supports light and heavy isotope labeling at the cellular level and improves signal-to-noise ratio via MS2 quantitative reporter ions—without increasing spectral complexity.
Using this novel cross-linker, the researchers successfully mapped the protein interaction network in primary neurons derived from a single-embryo mouse. They also achieved quantitative detection of transient and weak interactions within dynamic liquid-liquid phase separation environments.
"By addressing key limitations in in vivo cross-linking proteomics, DPST provides a powerful solution for both qualitative and quantitative XL-MS analysis, with strong potential to drive advances in biomedical research and drug discovery," said Prof. ZHANG.