Mass spectrometry method fixes short-lived protein interactions in place

While many protein interactions are detectable with current methods, others are too dynamic and transient to be reliably captured. To address this limitation, researchers created a proteomics workflow called PhIX-MS (Photo-induced In situ Crosslinking Mass Spectrometry). PhIX-MS allows scientists to stabilize, or fix in place, weak or short-lived protein interactions before purifying them for further analysis. Activation takes just nanoseconds, trapping only natively interacting proteins and minimizing false positives. Researchers then use mass spectrometry to identify exactly which protein regions were chemically linked, revealing their architecture inside the cell. The researchers demonstrated the power of PhIX-MS by examining the proteasome, the cellular machine that degrades proteins. By incorporating insights from cryo-electron microscopy and AlphaFold predictions, they revealed the structural context underlying key regulators of proteasome activity. PhIX-MS is broadly applicable and has the capacity to reveal the dynamic landscape of a wide range of key protein assemblies.

“This approach lets us zoom in on one molecular machine at a time,” said co-lead author Francis O’Reilly, PhD, Department of Structural Biology. “We can crosslink its parts together inside the cell, identify the connections by mass spectrometry and use those contacts to understand how the machine is built and works.”

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