Characterization of S-palmitoylation dependent protein localization by hyperplexed spatial proteomics
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S-Palmitoylation is a post translational modification (PTM) associated with numerous cancers and neurological diseases. It is a reversible type of lipidation defined by the addition of a palmitic acid to a cysteine residue. Regulation of this PTM depends on the activity of palmitoyl S-acyl transferases and palmitoyl thioesterases which add and remove the palmitoyl moiety, respectively. S-palmitoylation can have various effects on proteins, of which a change in intracellular localization is most substantiated. While methods for large scale identification of palmitoylated proteins have become well established, the characterization of S-palmitoylation dependent translocations has so far only been studied on a single protein basis. This study aimed to identify S-palmitoylation dependent translocating proteins. The broad spectrum depalmitoylase inhibitor Palmostatin B (PalmB) was employed in combination with differential centrifugation based spatial proteomics to determine translocating proteins. In addition, SILAC-TMT hyperplexing was used to allow for the combined quantitation of treatment groups and spatial proteomics fractions. Using this method we identified 48 potentially translocating proteins. A high overrepresentation of S-palmitoylated proteins was observed among the translocators indicating that the translocation of these enriched proteins may have occurred in response to a change in palmitoylation. Additionally, a STRING database search found many interacting proteins within this group suggesting that some translocations may occur as a result of the translocation of a binding partner. We demonstrate that spatial proteomics can be used as a hypothesis-generating method to identify potential S-palmitoylation dependent translocating proteins. These potential translocators can be used as a starting point for further study into S-palmitoylation dependent protein localization.