Robert Arkowitz (@robertarkowitz.bsky.social)

Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells The function of a protein is determined by its structure, which may change dynamically in response to post-translational modifications, interaction with other molecules, or environmental factors like temperature. Limited proteolysis-coupled mass spectrometry (LiP-MS) captures such structural alterations on a proteome-wide scale via the detection of altered protease susceptibility patterns of proteins. However, this technique has so far required cell lysis, which exposes proteins to non-native conditions and can disrupt labile interactions such as those occurring within biomolecular condensates. To study protein structures directly within cells, we developed in-cell LiP-MS. We optimized conditions for introduction of proteinase K into human cells using electroporation and validated that intracellular cleavage occurs. In-cell LiP-MS captured not only the known binding of rapamycin to FKBP1A within the cell, but also downstream effects of pathway activation. Moreover, it detected global protein structural alterations upon sodium arsenite treatment and captured the structural dynamics of hundreds of proteins from biomolecular condensates with peptide level resolution and within live human cells. We detected known and novel structural alterations of proteins from stress granules as well as from nuclear speckles and validated alteration of nuclear speckles by fluorescence microscopy. Our dataset provides a resource describing the structural changes of human proteins in response to cellular stress and pinpoints structurally altered regions. Further, comparison of LiP-based structural fingerprints before and after cell lysis revealed which human proteins are susceptible to structural change upon cell lysis, therefore guiding the design of future experiments requiring native protein structures. ### Competing Interest Statement P.P. is a scientific advisor for the company Biognosys AG (Zurich, Switzerland) and an inventor of a patent licensed by Biognosys AG that covers the LiP-MS method used in this work. F.E., R.F., and P.P. have filed a patent application based on this work (European Patent Application No. 23197690). The remaining authors declare no competing interests. https://www.biorxiv.org/content/10.1101/2025.02.21.639445v1