An enzymatic cascade enables sensitive and specific proximity labeling proteomics in challenging biological systems

Ascorbate peroxidase (APEX) is a proximity labeling enzyme used for subcellular proteomics at high spatial and temporal resolution. However, toxicity of its substrate hydrogen peroxide and background labeling by endogenous peroxidases limit its use to in vitro studies of specific cell types. To minimize toxicity and reduce non-specific background labeling we establish a more versatile in situ APEX activation (iAPEX) workflow by combining APEX2 with a D-amino acid oxidase to locally produce hydrogen peroxide. Using iAPEX, we profile the proteomes of a cellular microdomain, the primary cilium, in cell lines not readily accessible to conventional APEX labeling and identify unknown ciliary proteins. Our study validates common ciliary proteins across two distinct cell lines, while observed differences may reflect heterogeneity in primary cilia proteomes. Furthermore, iAPEX proximity labeling is applicable to a range of cellular compartments including mitochondria and lipid droplets and can be employed in Xenopus laevis, which provides a proof-of-concept for future in vivo applications.

Authors

Institutions

Publication Details

Journal
Nature Communications
Published
2025-11-03
DOI
https://doi.org/10.1038/s41467-025-65405-8
Citations
2
Primary Topic
Biotin and Related Studies
Type
article
Field-Weighted Citation Impact
2.30
Controls
|||
ALL TIME
JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
article

An enzymatic cascade enables sensitive and specific proximity labeling proteomics in challenging biological systems

Tommy J Sroka, Karina von der Malsburg, Bianca Schrul, Peter Walentek et al.
2 citations
Nature Communications
Biotin and Related Studies
2.30
article

An enzymatic cascade enables sensitive and specific proximity labeling proteomics in challenging biological systems

Tommy J Sroka, Karina von der Malsburg, Bianca Schrul, Peter Walentek, Josefine Hoeren, Dominik Weber, Valentina Trivigno, David U. Mick, Avishek Prasai, Daniela Yildiz, Kerstin Feistel, Per Haberkant, Lea K. Sanwald, Valerie Chaumet, Louisa Magdalena Krauß
article en
2 citations

Abstract

Ascorbate peroxidase (APEX) is a proximity labeling enzyme used for subcellular proteomics at high spatial and temporal resolution. However, toxicity of its substrate hydrogen peroxide and background labeling by endogenous peroxidases limit its use to in vitro studies of specific cell types. To minimize toxicity and reduce non-specific background labeling we establish a more versatile in situ APEX activation (iAPEX) workflow by combining APEX2 with a D-amino acid oxidase to locally produce hydrogen peroxide. Using iAPEX, we profile the proteomes of a cellular microdomain, the primary cilium, in cell lines not readily accessible to conventional APEX labeling and identify unknown ciliary proteins. Our study validates common ciliary proteins across two distinct cell lines, while observed differences may reflect heterogeneity in primary cilia proteomes. Furthermore, iAPEX proximity labeling is applicable to a range of cellular compartments including mitochondria and lipid droplets and can be employed in Xenopus laevis, which provides a proof-of-concept for future in vivo applications.

Nature CommunicationsVol. 16(1)
University of Hohenheim (DE), University of Freiburg (DE), Institut für Sozialforschung und Sozialwirtschaft (DE), Hochschule der Bildenden Künste Saar (DE), Saarland University (DE)
Openalex Percentile: Top 11%
Biotin and Related Studies
2.30
DOIOpen Access PDFOpenAlex
AI Navigator

Ask Laika to Summarize, Analyze, and Connect papers live on the map.

Summarize Papers & Methodologies

Extract key findings, datasets, and comparative methods across publications.

Benchmark Rankings & Visual Analytics

Rank top research institutions, authors, funders, topics, and journals by Field-Weighted Citation Impact (FWCI) and paper volume with instant charts.

Connect Distant Disciplines

Bridge topological clusters on the map to find hidden collaborative intersections.