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Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

ABSTRACT: To study protein structures directly within cells, we developed in-cell limited proteolysis-coupled mass spectrometry (in-cell LiP-MS). Conditions for introduction of proteinase K into human cells using electroporation were optimized and validated for intracellular cleavages. In-cell LiP-MS captured the known binding of rapamycin to FKBP1A within the cell and detected global protein structural alterations upon sodium arsenite treatment. It captured the structural dynamics of hundreds of proteins from biomolecular condensates with peptide level resolution and within live human cells such as structural alterations of individual sites on the involved proteins, such as known RNA-binding and intrinsically-disordered regions, and dissected the timing of the different events. 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 arsenite 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ludovic Gillet

LAB HEAD: Paola Picotti

PROVIDER: PXD069095 | Pride | 2025-11-09

REPOSITORIES: Pride

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			Action	DRS
	20230420_105758_FE502_230419_time_Report.csv	Csv
	20230424_154617_230424_FE504_EP_Report.csv	Csv
	230327_EC1_elsaesfr_FE502_001.raw	Raw
	230327_EC1_elsaesfr_FE502_002.raw	Raw
	230327_EC1_elsaesfr_FE502_003.raw	Raw

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Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

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