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

• Published in: Angewandte Chemie International Edition Vol. 59; no. 37; pp. 16069 - 16075
• Main Authors: Chen, Nan, Liu, Yuan, Li, Yuanpei, Wang, Chu
• Format: Journal Article
• Language: English
• Published: WEINHEIM Wiley 07.09.2020; Wiley Subscription Services, Inc
• Edition: International ed. in English
• Subjects: Alanine; Animals; Biosynthesis; chemical biology; Chemistry; Chemistry, Multidisciplinary; Eukaryotes; Fatty acids; Folic acid; Humans; Labeling; Mammalian cells; Mammals; mass spectrometry; Mass Spectrometry - methods; metabolic labelling; Metabolism; Mitochondria; Pantetheine - analogs & derivatives; Pantetheine - metabolism; phosphopantetheinylation; Physical Sciences; Prokaryotes; protein modifications; Protein Processing, Post-Translational; Proteins; Proteomics; Proteomics - methods; Reductases; Science & Technology; Substrates; ACYL CARRIER PROTEIN; protein modifications; chemical biology; phosphopantetheinylation; PROSTHETIC GROUP; mass spectrometry; SYNTHETASE; metabolic labelling; COENZYME-A; IDENTIFICATION; MASS-SPECTROMETRY; PEPTIDE
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202004105

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). By developing a new generation of pantetheine analogue probes, the metabolic labelling and chemoproteomic profiling of protein 4′‐phosphopantetheinylation in mammalian cells were achieved. Targeted chemical proteomics facilitated the identification of exact 4′‐phosphopantetheinylation sites, including all five currently known sites and a potential site in the mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).