Chemical Proteomic Profiling of Protein 4′‐Phosphopantetheinylation in Mammalian Cells

Abstract Protein 4′‐phosphopantetheinylation is an essential post‐translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as β‐alanine activation. To explore existing and new substrates of 4′‐phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4′‐phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4′‐phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4′‐phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).