PXD060753

PXD060753 is an original dataset announced via ProteomeXchange.

Title	Directed Evolution of APOX for Proximity Labeling Using Electron-Deficient Phenols
Description	Proximity labeling identifies endogenous proteins in specific subcellular regions. APEX2 is an enzyme that enables PL with high temporal resolution by converting biotin phenol (BP) into a radical that tags nearby proteins. However, the BP radical has a relatively large diffusion radius, limiting spatial resolution. Replacing BP with an electron-deficient nitrophenol (NP) probe could potentially improve the spatial resolution by generating more reactive radicals, but APEX2 cannot efficiently activate electron-deficient phenols. Here, we report the directed evolution of APOX, a quadruple mutant of APEX2 with a higher reduction potential, exhibiting 6-fold and 2.5-fold faster oxidation of NPs and unsubstituted phenols, respectively. Using APOX with a membrane-permeable alkyne-NP probe, we demonstrate PL in living mammalian cells, including proteomic mapping with excellent subcellular-compartment specificity. APOX expands heme peroxidase PL by accessing electron-deficient probes, opening opportunities to further tune the diffusion radius and enhance the tagging of biomolecules beyond proteins.
HostingRepository	PRIDE
AnnounceDate	2026-04-10
AnnouncementXML	Submission_2026-04-09_18:36:52.220.xml
DigitalObjectIdentifier	https://doi.org/10.6019/PXD060753
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Alexander Solivais
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606; scientific name: Bos taurus (Bovine); NCBI TaxID: NEWT:9913;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF; Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-02-12 21:17:37	ID requested
⏵ 1	2026-04-09 18:36:52	announced

10.6019/PXD060753;

10.1016/J.CHEMBIOL.2026.03.011;

submitter keyword: lfq, labeling, label-free quantification,APEX, biotin

Lloyd M Smith
contact affiliation	University of Wisconsin-Madison, Department of Chemistry
contact email	smith@chem.wisc.edu
lab head
Alexander Solivais
contact affiliation	University of Wisconsin-Madison
contact email	alexsolivais@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD060753
     1. Label: PRIDE project
     2. Name: Directed Evolution of APOX for Proximity Labeling Using Electron-Deficient Phenols