Harnessing Collective Electrophilic Reactivity of Affinity Labeling Probes for Versatile Target Identification

Affinity labeling has been employed as a powerful tool for target protein identification, yet outcomes are often unpredictable due to the inherent protein-dependent reactivity of electrophilic labels. This problem is especially pronounced for carbohydrate ligands with weak binding affinity. We report a data-driven approach to designing affinity labeling probes that addresses the inherent limitations of traditional single-electrophile probes. Using the lactose-lectin pairs as model systems, we developed a multiwell plate reaction analysis method to quantitatively evaluate a library of gold nanoparticle-based electrophile probes. By treating the electrophile library as a multisensor array, we characterized target proteins based on their unique reactivity profiles. Application of linear discriminant analysis (LDA) to the reactivity data set revealed that proteins can be distinguished by their reactivity fingerprints. Optimization using LDA identified a minimal subset of five electrophiles spanning distinct reaction mechanisms, which is capable of efficient protein discrimination. Guided by these multivariate analyses, we developed the heteromultivalent gold-nanoparticle probe that simultaneously displays the optimized electrophile ensemble. Our five-electrophile probe achieved higher labeling efficiency for a target protein in complex lysate compared to an equivalent mixture of five single-electrophile probes. Our findings provide a systematic platform for the design of versatile affinity labels, offering an effective new strategy for capturing elusive target proteins.