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Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

Nature Protocols volume 10, pages 134–148 (2015)Cite this article

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Abstract

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions.

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Figure 1: Schematic of HF5; separation principle and protocol workflow.
Figure 2: HF5 separation device setup and flow parameters.
Figure 3: Results: carbonyl content and SDS-PAGE of protein aggregates.
Figure 4: Results: Separation and characterization of protein aggregates.

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Acknowledgements

We thank Superon for the loan of the Eclipse DUALTEC flow FFF separation system and Eclipse ISIS software, and Wyatt Technology for the loan of the MALS detector DAWN EOS and Astra software. We thank C. Johann for the valuable suggestions and comments on the manuscript and S. Elsenberg (Superon) for technical assistance. The work was supported by National Institute on Aging (NIA) grant PO1AG031781 to A.M.C. and PO1AG031782 to L.S. V.Z. is supported by the PhD program in Genetics and Cell Biology at the University of Tuscia, Department of Ecology and Biology (DEB).

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Authors and Affiliations

  1. Department of Pathology, Albert Einstein College of Medicine, New York, USA

    Maya Tanase, Valerio Zolla, Cristina C Clement, Francesco Borghi, Aleksandra M Urbanska & Laura Santambrogio

  2. Department of Chemistry, University of Bologna, Bologna, Italy

    Maya Tanase, Francesco Borghi, Barbara Roda, Andrea Zattoni & Pierluigi Reschiglian

  3. Department of Developmental Molecular Biology, Albert Einstein College of Medicine, New York, USA

    Jose Antonio Rodriguez-Navarro & Ana Maria Cuervo

  4. byFlow SRL, Bologna, Italy

    Barbara Roda, Andrea Zattoni & Pierluigi Reschiglian

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Contributions

M.T., B.R., A.Z., P.R., A.M.C. and L.S. designed the experiments; M.T., V.Z., C.C.C., F.B., A.M.U. and J.A.R.-N. performed the experiments; and M.T., V.Z., C.C.C., B.R., A.Z., P.R., A.M.C. and L.S. wrote the paper.

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Correspondence to Laura Santambrogio.

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Competing interests

A.Z., B.R. and P.R. are associates of the academic spinoff company byFlow SRL. The company mission includes know-how transfer, development, and application of novel technologies and methodologies for the analysis and characterization of samples of nano-biotechnological interest.

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Tanase, M., Zolla, V., Clement, C. et al. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates. Nat Protoc 10, 134–148 (2015). https://doi.org/10.1038/nprot.2015.009

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