Background/aim: Protein palmitoylation is a reversible lipid modification that plays a crucial role in regulating the localization and function of membrane-associated proteins. The modification has been shown to affect protein-protein interactions, membrane trafficking, and signaling pathways.SILAC-based proteomic analysis and the process of cell division to study the role of palmitoylation and enzymes that transfer palmitoyl groups during cell division.
Methods: To prepare the cells for analysis, we synchronized HeLa cells at two key cell cycle stages: interphase and mitosis, using a well-established protocol.SILAC labeling was applied to create "heavy" and "light" cell populations, where heavy cells were metabolically labeled with 17-ODYA and light cells with palmitic acid, and vice versa in biological replicates.A clickable palmitic acid analog, 17-ODYA (17-octadecynoic acid), is incorporated into palmitoylation sites through a metabolic labeling method.Subsequently, the cells are lysed, and the click reaction is performed using biotin azide or rhodamine azide.The cells were cultured in DMEM medium devoid of lysine and arginine amino acids, supplemented with isotopically labeled lysine and arginine for heavy labeling and natural isotopes for light labeling. Subsequently, we performed click chemistry on the lysed SILAC-labeled cells, followed by biotinylation of palmitoylated proteins using streptavidin-coated beads. After enrichment, proteins were subjected to trypsin digestion, and resulting peptides were analyzed using mass spectrometry. The acquired data were processed using MaxQuant and Proteome Discoverer software against the Swiss-Prot human proteome database.
Results and conclusions:
As our prilimenery data using SILAC-labeled heavy and light samples, we have identified proteins with significantly altered SILAC ratios in samples labeled with 17-ODYA and palmitic acid. Upon further analysis of these proteins in the SwissPalm database, it was observed that the palmitoylated protein ratios exhibited a substantial increase. These findings confirm the successful identification of palmitoylated proteins. This data provides insights into the impact of palmitoylation on mitosis.