title: Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates.
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.
authors.affiliation: 1] Department of Pathology, Albert Einstein College of Medicine, New York, USA. [2] Department of Chemistry, University of Bologna, Bologna, Italy.
authors.forename: Maya
authors.initials: M
authors.lastname: Tanase
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Valerio
authors.initials: V
authors.lastname: Zolla
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Cristina C
authors.initials: CC
authors.lastname: Clement
authors.affiliation: 1] Department of Pathology, Albert Einstein College of Medicine, New York, USA. [2] Department of Chemistry, University of Bologna, Bologna, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Borghi
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Aleksandra M
authors.initials: AM
authors.lastname: Urbanska
authors.affiliation: Department of Developmental Molecular Biology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Rodriguez-Navarro
authors.affiliation: 1] Department of Chemistry, University of Bologna, Bologna, Italy. [2] byFlow SRL, Bologna, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Roda
authors.affiliation: 1] Department of Chemistry, University of Bologna, Bologna, Italy. [2] byFlow SRL, Bologna, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Zattoni
authors.affiliation: 1] Department of Chemistry, University of Bologna, Bologna, Italy. [2] byFlow SRL, Bologna, Italy.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Reschiglian
authors.affiliation: Department of Developmental Molecular Biology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Cuervo
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Santambrogio
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002850:Chromatography, Gel; D057446:Hydrodynamics; D008027:Light; D010269:Paraquat; D066329:Protein Aggregates / Q000235:genetics*; D012542:Scattering, Radiation; D014461:Ultracentrifugation
chemical_list: D066329:Protein Aggregates; D010269:Paraquat