Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample
Protein S-palmitoylation is the process by which a palmitoyl fatty acid is attached to a cysteine residue of a protein via a thioester bond. A range of methodologies are available for the detection of protein S-palmitoylation. In this study, two methods for the S-palmitoylation of different proteins...
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2025-01-01
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| author | Nadine Merz Karin Schilling Dominique Thomas Lisa Hahnefeld Sabine Grösch |
| author_facet | Nadine Merz Karin Schilling Dominique Thomas Lisa Hahnefeld Sabine Grösch |
| author_sort | Nadine Merz |
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| description | Protein S-palmitoylation is the process by which a palmitoyl fatty acid is attached to a cysteine residue of a protein via a thioester bond. A range of methodologies are available for the detection of protein S-palmitoylation. In this study, two methods for the S-palmitoylation of different proteins were compared after metabolic labeling of cells with 15-hexadecynoic acid (15-YNE) to ascertain their relative usefulness. It was hypothesized that labeling cells with a traceable lipid would affect lipid metabolism and the cellular lipidome. In this study, we developed a method to track 15-YNE incorporation into lipids using liquid chromatography high-resolution mass spectrometry (LC-HRMS) as well as protein palmitoylation in the same sample. We observed a time- and concentration-dependent S-palmitoylation of calnexin and succinate dehydrogenase complex flavoprotein subunit A (SDHA) depending on the cell type. The detection of S-palmitoylation with a clickable fluorophore or biotin azide followed by immunoprecipitation is shown to be equally useful. 15-YNE was observed to be incorporated into a wide array of lipid classes during the process, yet it did not appear to modify the overall lipid composition of the cells. In conclusion, we show that 15-YNE is a useful tracer to detect both protein S-palmitoylation and lipid metabolism in the same sample. |
| format | Article |
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| institution | Kabale University |
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| spelling | doaj-art-81dc747fe35643f69ea3c6d630d809ff2025-01-24T13:43:50ZengMDPI AGMolecules1420-30492025-01-0130237710.3390/molecules30020377Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same SampleNadine Merz0Karin Schilling1Dominique Thomas2Lisa Hahnefeld3Sabine Grösch4Goethe University Frankfurt, Institute of Clinical Pharmacology, Faculty of Medicine, Theodor Stern Kai 7, 60590 Frankfurt am Main, GermanyGoethe University Frankfurt, Institute of Clinical Pharmacology, Faculty of Medicine, Theodor Stern Kai 7, 60590 Frankfurt am Main, GermanyGoethe University Frankfurt, Institute of Clinical Pharmacology, Faculty of Medicine, Theodor Stern Kai 7, 60590 Frankfurt am Main, GermanyGoethe University Frankfurt, Institute of Clinical Pharmacology, Faculty of Medicine, Theodor Stern Kai 7, 60590 Frankfurt am Main, GermanyGoethe University Frankfurt, Institute of Clinical Pharmacology, Faculty of Medicine, Theodor Stern Kai 7, 60590 Frankfurt am Main, GermanyProtein S-palmitoylation is the process by which a palmitoyl fatty acid is attached to a cysteine residue of a protein via a thioester bond. A range of methodologies are available for the detection of protein S-palmitoylation. In this study, two methods for the S-palmitoylation of different proteins were compared after metabolic labeling of cells with 15-hexadecynoic acid (15-YNE) to ascertain their relative usefulness. It was hypothesized that labeling cells with a traceable lipid would affect lipid metabolism and the cellular lipidome. In this study, we developed a method to track 15-YNE incorporation into lipids using liquid chromatography high-resolution mass spectrometry (LC-HRMS) as well as protein palmitoylation in the same sample. We observed a time- and concentration-dependent S-palmitoylation of calnexin and succinate dehydrogenase complex flavoprotein subunit A (SDHA) depending on the cell type. The detection of S-palmitoylation with a clickable fluorophore or biotin azide followed by immunoprecipitation is shown to be equally useful. 15-YNE was observed to be incorporated into a wide array of lipid classes during the process, yet it did not appear to modify the overall lipid composition of the cells. In conclusion, we show that 15-YNE is a useful tracer to detect both protein S-palmitoylation and lipid metabolism in the same sample.https://www.mdpi.com/1420-3049/30/2/377Cy5.5-azidebiotinprotein palmitoylationalkyneclick reactionLinex |
| spellingShingle | Nadine Merz Karin Schilling Dominique Thomas Lisa Hahnefeld Sabine Grösch Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample Molecules Cy5.5-azide biotin protein palmitoylation alkyne click reaction Linex |
| title | Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample |
| title_full | Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample |
| title_fullStr | Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample |
| title_full_unstemmed | Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample |
| title_short | Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample |
| title_sort | cell labeling with 15 yne is useful for tracking protein palmitoylation and metabolic lipid flux in the same sample |
| topic | Cy5.5-azide biotin protein palmitoylation alkyne click reaction Linex |
| url | https://www.mdpi.com/1420-3049/30/2/377 |
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