Stoichiometry-Dependent Specificity in Biotin Enrichment: A Benchmarking Framework for Proximity-Labeling Proteomics.
- 1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, Utrecht3584CH, The Netherlands.
- 2Netherlands Proteomics Center, Padualaan 8, Utrecht3584CH, The Netherlands.
- 3Department of Cell Biology, Neurobiology and Biophysics, Utrecht University, Padualaan 8, Utrecht3584CH, The Netherlands.
Abstract
Proximity labeling methods (including BioID, TurboID, and ultraID), along with surface proteomics and microdomain mapping, enable proteome-wide identification of spatially proximal proteins via MS-based analysis. These workflows require specific enrichment of biotinylated proteins using affinity purification, yet enrichment specificity can often be compromised by nonspecifically bound proteins. As labeling strategies are increasingly applied to complex biological samples with low protein input or low biotin stoichiometry, accurately distinguishing true targets from the background becomes a major analytical challenge. Despite its critical impact on data quality and interpretation, the influence of the biotinylation level and protein input on enrichment performance remains poorly characterized, limiting the reliability of proximity labeling experiments. To address this, we established a quantitative benchmarking framework that systematically evaluates biotin enrichment under controlled conditions, including scenarios of low biotin stoichiometry. Using this setup, we show that enrichment specificity strongly depends on biotin stoichiometry: higher levels of biotinylation in samples yield high specificity, whereas low biotinylation increases nonspecific background. Reduced protein input further limits the recovery of true targets, yet maintains enrichment specificity, highlighting sensitivity constraints of enrichment-based workflows. We apply this framework to biotinylated extracellular vesicle (EV) cargo uptake in recipient cells using ultraID-CD63 labeling. Detection of the most abundant EV cargo proteins under low-biotinylation conditions indicates that current workflows approach the lower bounds of biotin enrichment sensitivity. Together, these standards provide a practical reference for evaluating and optimizing biotin enrichment workflows, supporting quantitative and reproducible proximity labeling in proteomics.
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BibTeX
@article{zala2026stoichiometrydependent,
title = {{Stoichiometry-Dependent Specificity in Biotin Enrichment: A Benchmarking Framework for Proximity-Labeling Proteomics.}},
author = {Christopher Árpád Zala and Maria Cristina Trueba Sanchez and Jelle van den Bor and Tijmen Willemsens and Frederik Johannes Verweij and Maarten Altelaar and Kelly E Stecker},
journal = {Analytical chemistry},
year = {2026},
month = sep,
doi = {10.1021/acs.analchem.6c03011},
url = {https://europepmc.org/article/MED/42915293},
note = {CC BY},
}
RIS
TY - JOUR
TI - Stoichiometry-Dependent Specificity in Biotin Enrichment: A Benchmarking Framework for Proximity-Labeling Proteomics.
AU - Christopher Árpád Zala
AU - Maria Cristina Trueba Sanchez
AU - Jelle van den Bor
AU - Tijmen Willemsens
AU - Frederik Johannes Verweij
AU - Maarten Altelaar
AU - Kelly E Stecker
PY - 2026
DA - 2026/09/01
JO - Analytical chemistry
DO - 10.1021/acs.analchem.6c03011
UR - https://europepmc.org/article/MED/42915293
AB - Proximity labeling methods (including BioID, TurboID, and ultraID), along with surface proteomics and microdomain mapping, enable proteome-wide identification of spatially proximal proteins via MS-based analysis. These workflows require specific enrichment of biotinylated proteins using affinity purification, yet enrichment specificity can often be compromised by nonspecifically bound proteins. As labeling strategies are increasingly applied to complex biological samples with low protein input or low biotin stoichiometry, accurately distinguishing true targets from the background becomes a major analytical challenge. Despite its critical impact on data quality and interpretation, the influence of the biotinylation level and protein input on enrichment performance remains poorly characterized, limiting the reliability of proximity labeling experiments. To address this, we established a quantitative benchmarking framework that systematically evaluates biotin enrichment under controlled conditions, including scenarios of low biotin stoichiometry. Using this setup, we show that enrichment specificity strongly depends on biotin stoichiometry: higher levels of biotinylation in samples yield high specificity, whereas low biotinylation increases nonspecific background. Reduced protein input further limits the recovery of true targets, yet maintains enrichment specificity, highlighting sensitivity constraints of enrichment-based workflows. We apply this framework to biotinylated extracellular vesicle (EV) cargo uptake in recipient cells using ultraID-CD63 labeling. Detection of the most abundant EV cargo proteins under low-biotinylation conditions indicates that current workflows approach the lower bounds of biotin enrichment sensitivity. Together, these standards provide a practical reference for evaluating and optimizing biotin enrichment workflows, supporting quantitative and reproducible proximity labeling in proteomics.
SN - 0003-2700
SN - 1520-6882
ER -