In a recent study published in Nature Communications (Michael et al., 2024), researchers developed an advanced crosslinking mass spectrometry (XL-MS) technique that utilizes formaldehyde-based cell fixation to enhance the mapping of protein-protein interactions. Traditional in vivo XL-MS faces challenges with low cell permeability and long reaction times, often causing distortions in cellular structures and unreliable data on protein interactions. The study addresses these limitations by stabilizing cells with a 4% formaldehyde fixation prior to the crosslinking process, preserving ultrastructure and enhancing crosslinking effectiveness.
The researchers first grew A549 lung cancer cells and then applied formaldehyde for rapid fixation. The fixed cells underwent gas chromatography-mass spectrometry (GC-MS) analysis, revealing that the fixation did not interfere with amine-reactive crosslinkers. This process uncoupled cellular movement from crosslinking, allowing thorough labeling with common NHS-based crosslinkers. With this approach, the researchers mapped high-resolution protein interactions without the cellular damage typically associated with standard XL-MS methods.
Experiments using disuccinimidyl suberate (DSS), a common crosslinker, on both fixed and permeabilized cells, yielded higher crosslinking efficiency than live-cell treatments. Fixed cells exhibited almost double the number of crosslinked peptides, demonstrating the fixation’s role in stabilizing and exposing cellular proteins. Further, the formaldehyde fixation did not compete with amine-specific crosslinking reagents, allowing the use of a broader range of crosslinkers.
This method proves promising for cellular research as it preserves cellular integrity and reduces background interference, making it suitable for in-depth studies of protein-protein interactions in their native spatial context. The study suggests that formaldehyde fixation can become a powerful tool in proteomics, enabling precise interaction mapping and aiding our understanding of cellular dynamics without the structural compromises seen in traditional XL-MS techniques.
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Reference
Michael, A.R.M., Amaral, B.C., Ball, K.L. et al. Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure. Nat Commun 15, 8537 (2024). https://doi.org/10.1038/s41467-024-52844-y
