Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity

Abstract

The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a ‘latent’ analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.

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Figure 1: Latent labelling enables the development of native microfluidic analysis systems.
Figure 2: Precise control of reaction time enables quantitative, fluorogenic protein labelling before detection.
Figure 3: Sizing proteins, heterogeneous mixtures and protein complexes.

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Acknowledgements

The authors acknowledge the European Research Council, Biotechnology and Biological Sciences Research Council, Wellcome Trust, Newman Foundation, Winston Churchill Foundation and Elan Pharmaceuticals for financial support. E.D.G. was supported by the Medical Research Council (G1002272). The authors thank J. Steyaert at the Free University of Brussels for sharing the NbSyn1a clone.

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T.P.J.K. and C.M.D. supervised the research. E.V.Y., L.R., M.V., C.M.D. and T.P.J.K. conceived and designed the experiments. E.V.Y. performed the experiments. E.V.Y. and T.M. analysed the data. E.J.D.G., P.A. and S.L. contributed materials and/or analysis tools. E.V.Y., C.M.D. and T.P.J.K. wrote the paper, and all authors commented on the paper.

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Correspondence to Christopher M. Dobson or Tuomas P. J. Knowles.

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Part of the work described here has been the subject of a patent application filed by Cambridge Enterprise Ltd, a fully owned subsidiary of the University of Cambridge (now licensed to Fluidic Analytics, of which C.M.D. is a scientific advisor and T.P.J.K. is a board member).

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Yates, E., Müller, T., Rajah, L. et al. Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity. Nature Chem 7, 802–809 (2015). https://doi.org/10.1038/nchem.2344

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