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

Nature Chemistry volume 7, pages 802–809 (2015)Cite this article

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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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References

  1. Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    CAS  PubMed  Google Scholar 

  2. Suh, E. H. et al. Stilbene vinyl sulfonamides as fluorogenic sensors of and traceless covalent kinetic stabilizers of transthyretin that prevent amyloidogenesis. J. Am. Chem. Soc. 135, 17869–17880 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Aguzzi, A. & Calella, A. M. Prions: protein aggregation and infectious diseases. Phys. Rev. 89, 1105–1152 (2009).

    CAS  Google Scholar 

  4. Campioni, S. et al. The presence of an air–water interface affects formation and elongation of α-synuclein fibrils. J. Am. Chem. Soc. 136, 2866–2875 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Adamcik, J. et al. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nature Nanotech. 5, 423–428 (2010).

    Article  CAS  Google Scholar 

  6. Chiti, F. & Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Arkin, M. R. & Whitty, A. The road less traveled: modulating signal transduction enzymes by inhibiting their protein–protein interactions. Curr. Opin. Chem. Biol. 13, 284–290 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Wells, J. A. & McClendon, C. L. Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature 450, 1001–1009 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Berggård, T., Linse, S. & James, P. Methods for the detection and analysis of protein–protein interactions. Proteomics 7, 2833–2842 (2007).

    Article  PubMed  Google Scholar 

  10. Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Yu, X., Xu, D. & Cheng, Q. Label-free detection methods for protein microarrays. Proteomics 6, 5493–5503 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Zuiderweg, E. R. P. Mapping protein–protein interactions in solution by NMR spectroscopy. Biochemistry 41, 1–7 (2001).

    Article  Google Scholar 

  14. Hanlon, A. D., Larkin, M. I. & Reddick, R. M. Free-solution, label-free protein–protein interactions characterized by dynamic light scattering. Biophys. J. 98, 297–304 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee, T. T. & Yeung, E. S. High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis. J. Chromatogr. A 595, 319–325 (1992).

    Article  CAS  Google Scholar 

  16. Bornhop, D. J. et al. Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science 317, 1732–1736 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Jungbauer, L. M., Yu, C., Laxton, K. J. & LaDu, M. J. Preparation of fluorescently-labeled amyloid-beta peptide assemblies: the effect of fluorophore conjugation on structure and function. J. Mol. Recogn. 22, 403–413 (2009).

    Article  CAS  Google Scholar 

  18. Herling, T. W. et al. Integration and characterization of solid wall electrodes in microfluidic devices fabricated in a single photolithography step. Appl. Phys. Lett. 102, 184102–4 (2013).

    Article  Google Scholar 

  19. Bruus, H. Theoretical Microfluidics (Oxford Master Series in Physics, Oxford Univ. Press, 2008).

    Google Scholar 

  20. Brody, J. P. & Yager, P. Diffusion-based extraction in a microfabricated device. Sens. Actuat. A 58, 13–18 (1997).

    Article  CAS  Google Scholar 

  21. Weigl, B. H. & Yager, P. Microfluidic diffusion-based separation and detection. Science 283, 346–347 (1999).

    Article  Google Scholar 

  22. Kamholz, A. E., Weigl, B. H., Finlayson, B. A. & Yager, P. Quantitative analysis of molecular interaction in a microfluidic channel: the T-sensor. Anal. Chem. 71, 5340–5347 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Hatch, A. et al. A rapid diffusion immunoassay in a T-sensor. Nature Biotechol. 19, 461–465 (2001).

    Article  CAS  Google Scholar 

  24. Kamholz, A. E., Schilling, E. A. & Yager, P. Optical measurement of transverse molecular diffusion in a microchannel. Biophys. J. 80, 1967–1972 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Knowles, T., Devlin, G., Dobson, C. & Welland, M. in Methods in Molecular Biology Vol. 752 (eds Hill, A. F., Barnham, K. J., Bottomley, S. P. & Cappai, R.) 137–145 (Humana, 2011).

    Google Scholar 

  26. Gao, D., Liu, H., Jiang, Y. & Lin, J.-M. Recent advances in microfluidics combined with mass spectrometry: technologies and applications. Lab Chip 13, 3309–3322 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Roth, M. Fluorescence reaction for amino acids. Anal. Chem. 43, 880–882 (1971).

    Article  CAS  PubMed  Google Scholar 

  28. Benson, J. R. & Hare, P. o-Phthalaldehyde fluorogenic detection of primary amines in the picomole range. comparison with fluorescamine and ninhydrin. Proc. Natl Acad. Sci. USA 72, 619–622 (1975).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Simons, S. S. & Johnson, D. F. The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines. J, Am. Chem. Soc. 98, 7098–7099 (1976).

    Article  CAS  Google Scholar 

  30. Sternson, L. A., Stobaugh, J. F. & Repta, A. J. Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents. Anal. Biochem. 144, 233–246 (1985).

    Article  CAS  PubMed  Google Scholar 

  31. Jacobson, S. C., Koutny, L. B., Hergenroeder, R., Moore, A. W. & Ramsey, J. M. Microchip capillary electrophoresis with an integrated postcolumn reactor. Anal. Chem. 66, 3472–3476 (1994).

    Article  CAS  Google Scholar 

  32. Otzen, D. Protein–surfactant interactions: a tale of many states. Biochim. Biophys. Acta 1814, 562–591 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Stobaugh, J., Repta, A., Sternson, L. & Garren, K. Factors affecting the stability of fluorescent isoindoles derived from reaction of o-phthalaldehyde and hydroxyalkylthiols with primary amines. Anal. Biochem. 135, 495–504 (1983).

    Article  CAS  PubMed  Google Scholar 

  34. Lindroth, P. & Mopper, K. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal. Chem. 51, 1667–1674 (1979).

    Article  CAS  Google Scholar 

  35. Svedas, V. J., Galaev, I. J., Borisov, I. L. & Berezin, I. V. The interaction of amino acids with o-phthalaldehyde: a kinetic study and spectrophometric assay of the reaction product. Anal. Biochem. 101, 188–195 (1980).

    Article  CAS  PubMed  Google Scholar 

  36. Horrocks, M. H. et al. Single-molecule measurements of transient biomolecular complexes through microfluidic dilution. Anal. Chem. 85, 6855–6859 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Waters, J. C. Accuracy and precision in quantitative fluorescence microscopy. J. Cell Biol. 185, 1135–1148 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. De Genst, E. J. et al. Structure and properties of a complex of alpha-synuclein and a single-domain camelid antibody. J. Mol. Biol. 402, 326–343 (2010).

    Article  CAS  PubMed  Google Scholar 

  39. Jha, A. K., Colubri, A., Freed, K. F. & Sosnick, T. R. Statistical coil model of the unfolded state: resolving the reconciliation problem. Proc. Natl Acad. Sci. USA, 102, 13099–13104 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ulmer, T. S., Bax, A., Cole, N. B. & Nussbaum, R. L. Structure and dynamics of micelle-bound human α-synuclein. J. Biol. Chem. 280, 9595–9603 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Dedmon, M. M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. & Dobson, C. M. Mapping long-range interactions in α-Synuclein using spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem. Soc. 127, 476–477 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Morar, A. S., Olteanu, A., Young, G. B. & Pielak, G. J. Solvent-induced collapse of α-synuclein and acid-denatured cytochrome c. Prot. Sci. 10, 2195–2199 (2001).

    Article  CAS  Google Scholar 

  43. Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. & Lansbury, P. T. NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry 35, 13709–13715 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Lord, R. S., Gubensek, F. & Rupley, J. A. Insulin self-association. Spectrum changes and thermodynamics. Biochemistry 12, 4385–4392 (1973).

    Article  CAS  PubMed  Google Scholar 

  45. Bocian, W. et al. Structure of human insulin monomer in water/acetonitrile solution. J. Biomol. NMR 40, 55–64 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Lin, M. & Larive, C. Detection of insulin aggregates with pulsed-field gradient nuclear magnetic resonance spectroscopy. Anal. Biochem. 229, 214–220 (1995).

    Article  CAS  PubMed  Google Scholar 

  47. Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. De Genst, E., Messer, A. & Dobson, C. M. Antibodies and protein misfolding: from structural research tools to therapeutic strategies. Biochim. Biophys. Acta 1844, 1907–1919 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. De Genst, E. & Dobson, C. M. in Methods in Molecular Biology Vol. 911 (eds Saerens, D. & Mulydermans, S.), 533–558 (2012).

    Article  CAS  PubMed  Google Scholar 

  50. Guilliams, T. et al. Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages. J. Mol. Biol. 425, 2397–2411 (2013).

    Article  CAS  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK

    Emma V. Yates, Thomas Müller, Luke Rajah, Erwin J. De Genst, Paolo Arosio, Michele Vendruscolo, Christopher M. Dobson & Tuomas P. J. Knowles

  2. Department of Biochemistry and Structural Biology, Lund University, Lund, SE221 00, Sweden

    Sara Linse

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Contributions

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.

Corresponding authors

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

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Competing interests

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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