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Here we introduce the third\u2010generation covalent TMP\u2010tag (TMP\u2010tag3) and showcase its comparison with HaloTag and SNAP\u2010tag. TMP\u2010tag3 is based on a proximity\u2010induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)\u2013acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP\u2010tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18\u2010kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal N\u2212C termini of eDHFR also enable facile insertion into various protein loops. TMP\u2010tag3, HaloTag, and SNAP\u2010tag are orthogonal to each other, collectively forming a toolbox for multiplexed live\u2010cell imaging of cellular proteins under fluorescence nanoscopy.<\/jats:p>","DOI":"10.1002\/anie.202207905","type":"journal-article","created":{"date-parts":[[2022,7,11]],"date-time":"2022-07-11T09:39:21Z","timestamp":1657532361000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":42,"title":["Third\u2010Generation Covalent TMP\u2010Tag for Fast Labeling and Multiplexed Imaging of Cellular Proteins"],"prefix":"10.1002","volume":"61","author":[{"given":"Jiaming","family":"Mo","sequence":"first","affiliation":[{"name":"National Biomedical Imaging Center Beijing Key Laboratory of Cardiometabolic Molecular Medicine College of Future Technology Peking University  Yiheyuan Road No.5 Beijing 100871 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