Hollow-Fiber Flow Field-Flow Fractionation for Mass Spectrometry: From Proteins to Whole Bacteria

Reschiglian, Pierluigi; Zattoni, Andrea; Rambaldi, Diana Cristina; Roda, Aldo; Moon, Myeong Hee

doi:10.1007/978-90-481-9815-3_2

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“…The HF5 method that was developed for this purpose was successful in fractionating and calculating the absolute MW of the separated species, the AvidinOX monomer, dimer and tetramer, proving that the protein oligomers were stable under both native and denaturing conditions. Over the past 20 years, HF5 was successfully used to separate protein mixtures 46,47 , including selected serum proteins under native conditions, and high- and low-density lipoproteins from human plasma samples 19,48–50 , as well as to enrich low-abundance high-density lipoproteins in blood serum 51,52 . HF5 was also used as a separation method for glycoproteins from bacterial lysates 53 and as a prefractionation method, in combination with complementary separation techniques such as capillary isoelectric focusing; it was also coupled on-line with MS for the proteomic analysis of bacteria ( Bacillus subtilis , Corynebacterium glutamicum and Escherichia coli ) 54 and human urinary proteins 55 .…”

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confidence: 99%

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

et al. 2014

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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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