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However, current enzymatic PL tools, such as TurboID and APEX2, are limited by their substrate structure and chemistry, which can generate significant background and\/or perturb cellular physiology. To address these limitations, we have developed aminoacyl-tRNA synthetase ID (aaRSID), a PL tool that leverages an engineered pyrrolysyl tRNA synthetase (PylRS) for proximity labeling of proteins. We chose PylRS because it can catalyze promiscuous lysine labeling in the absence of its cognate tRNA and utilize a variety of non-canonical amino acids (ncAAs) as substrates. Here, we demonstrate aaRSID\u2019s intrinsic proximity labeling activity, use directed evolution to improve this activity, and apply the improved mutant (aaRSID-Ma1.3) for subcellular proteomics and multiplexed imaging. Our work establishes aminoacyl-tRNA synthetases as a new PL enzyme class and introduces a versatile chemical platform for developing ncAA-derived probes to map cellular microenvironments, greatly expanding the applications possible of PL technology.<\/jats:p>","DOI":"10.64898\/2026.09.22.753581","type":"posted-content","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T17:15:18Z","timestamp":1790270118000},"source":"Crossref","is-referenced-by-count":0,"title":["aaRSID, an engineered pyrrolysyl-tRNA synthetase platform for multi-probe proximity proteomics"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-9231-2585","authenticated-orcid":false,"given":"Zhongjie","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Comparative Medicine, Yale University, School of Medicine, New Haven, CT 06520, 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