Hydrogel bead-based 3D multiplex platform for high precision and high throughput quantification of mTORC1-4E-BP1/2-dependent translation
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Published on: June 16, 2022
Mariana Ferreira Pissarra1, Lokman Alpsoy2, Malte Lauritz Breyer3
1Department Metabolism, Senescence and Autophagy, Research Center One Health Ruhr, University Alliance Ruhr & University Hospital Essen, University Duisburg-Essen, Essen, 45147, Germany; Center of Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, 45141, Germany; German Cancer Consortium (DKTK), Partner Site Essen/Duesseldorf, a Partnership Between German Cancer Research Center (DKFZ) and University Hospital Essen, Essen, 45147, Germany; Westdeutsches Tumorzentrum (WTZ), Essen, 45147, Germany; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, 6020, Austria; Freiburg Materials Research Center FMF, Albert-Ludwigs-University of Freiburg, Freiburg, 79104, Germany.
Background:
Quantifying protein phosphorylations and interactions across signaling networks is essential for quantitative biology and systems medicine. Current standard methods like immunoblotting offer limited throughput, precision, and scalability. We present an inverse-precision multiplex workflow based on a 3D hydrogel platform, enabling quantification of protein modifications and interactions within one assay. We chose the example of the mTORC1 (mTOR complex 1) kinase - 4E-BP1/2 signaling axis, clinically targeted by cancer therapies, which tunes protein biosynthesis to nutrient supply and stress.
Results:
We developed an inverse-precision design combining two orthogonal readouts, 4E-BP1/2 phosphorylation and eIF4E interaction. The design relies on two assays with inverse responses to mTORC1-dependent translation, offering inverse precision. Their combined analysis enhances the assay's robustness and dynamic range (LoD = 9% mTORC1 activity versus LoD = 15% for single readouts). Compared to conventional assays, 3D multiplexing increases precision by > fivefold (CV<4% versus 21% for immunoblotting around LoD), reduces sample consumption by 16-fold (7.8 μg protein input versus 125 μg in 2D format) and time effort by ≥ 80%, while offering a high degree of parallelization.
Significance:
Our workflow is compatible with small sample sizes, multiple technical and biological replicates, high-density dose-response curves and time-course data. We validated the assay for different physiological and experimental settings in cell culture and brain tissue. Standard microscopy equipment avoiding the need of specialized infrastructure, and assembly of custom antibody panels without dependence on fixed commercial kits present further advantages over established closed-panel systems. Its scalability, time-efficiency, and high precision make our platform a powerful tool for systems biology and precision medicine.
