Trueba Sanchez, M. C. | Search bioRxiv

bioRxiv2026

Stoichiometry-dependent specificity in biotin enrichment: a benchmarking framework for proximity labeling proteomics

Proximity labeling methods (including, BioID, TurboID, 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 non-specifically 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 background becomes a major analytical challenge. Despite its critical impact on data quality and interpretation, the influence of biotinylation level and protein input on enrichment performance remains poorly characterized, limiting the reliability of proximity labeling experiments. To address this, we establish 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 non-specific background. Reduced protein input further limits 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.

molecular biology↗

bioRxiv2024

Probing DNA damage sites reveals context-dependent and novel DNA damage response factors

DNA damage is a constant threat to genome integrity and function. Diminished capacity for DNA repair is linked to many human diseases, therefore understanding the molecular pathways responding to DNA damage is key for developing novel therapies. Lack of unbiased probes to report DNA damage dynamics and the associated proteins in living cells and animals limit our current efforts to completely understand DNA repair processes. In this study we overcome these limitations by engineering protein probes containing the tandem-BRCT domain of MCPH1, which we show to have a specific affinity for the DNA-damage-associated histone mark {gamma}H2AX. We employ these probes to track DNA damage dynamics in living cells exposed to a panel of different genotoxic insults and to visualize programmed double strand breaks during gametogenesis in living animals. We further utilize the binding selectivity of our probe to tether TurboID biotin ligases to chromatin and identify the DNA damage-associated proteome via proximity ligation. By comparing five different DNA damaging agents, we reveal the proteome associated with specific lesions, and identify multiple novel proteins with potential implications in damage response and repair. Among these novel proteins, we characterize the ubiquitin ligase UBE3A, the methyl-binding and proteasome-recruiting protein L3MBTL3, and the spliceosomal factor U2SURP, as previously uncharacterized effectors of DNA damage response. These functional datasets reveal the DNA damage-dependent proteomes and reveal novel insights into DNA damage response.

molecular biology↗