A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering.

Autor: Marassi V; Department of Chemistry G. Ciamician, University of Bologna, Via Selmi 2, 40126, Bologna, Italy; ByFlow Srl, Via dell'Arcoveggio 74, 40129, Bologna, Italy., Beretti F; Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Via del Pozzo, 71, 41124, Modena, Italy., Roda B; Department of Chemistry G. Ciamician, University of Bologna, Via Selmi 2, 40126, Bologna, Italy; ByFlow Srl, Via dell'Arcoveggio 74, 40129, Bologna, Italy. Electronic address: barbara.roda@unibo.it., Alessandrini A; CNR-Istituto Nanoscienze, S3, Via Campi 213/A, 41125, Modena, Italy; Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, Via Campi 213/A, 41125, Modena, Italy., Facci P; CNR-Istituto Nanoscienze, S3, Via Campi 213/A, 41125, Modena, Italy; Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, Via Campi 213/A, 41125, Modena, Italy., Maraldi T; Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Via del Pozzo, 71, 41124, Modena, Italy., Zattoni A; Department of Chemistry G. Ciamician, University of Bologna, Via Selmi 2, 40126, Bologna, Italy; ByFlow Srl, Via dell'Arcoveggio 74, 40129, Bologna, Italy., Reschiglian P; Department of Chemistry G. Ciamician, University of Bologna, Via Selmi 2, 40126, Bologna, Italy; ByFlow Srl, Via dell'Arcoveggio 74, 40129, Bologna, Italy., Portolani M; Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Via del Pozzo, 71, 41124, Modena, Italy.
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2019 Dec 09; Vol. 1087, pp. 121-130. Date of Electronic Publication: 2019 Aug 06.
DOI: 10.1016/j.aca.2019.08.003
Abstrakt: Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.
(Copyright © 2019 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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