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.

Adverse-Mode FFF: Multi-Force Ideal Retention Theory

“…In particular, the moments of the velocity distribution (including the first moment, which is the retention ratio) can be found. Doing so numerically verifies the analytical forms given by Equation (11), as seen in Figure 2.…”

“…The ideal moments of FFF for any number of external fields is given analytically by Equation (11). The ideal moments of traditional FFF with only a single external field are trivially obtained from Equation (11) by simply setting Γ = Λ −1rα .…”

“…Let us use Equation (11) to calculate the first three moments of velocity as a function of the multi-field device retention parameter Γ and solute sizer. The first moment is the retention ratio, which is given by:…”

“…Normal-mode FFF has proven to be a widely successful separation technique [1][2][3][4][5][6][7]. Recent applications of FFF include natural and artificial nanoparticles in environmental samples [8][9][10], protein aggregates [11], drug delivery nanocarriers [12], subcellular organelles [13], food applications [14], quantum dots [15], red blood cells [16], and many more. FFF has been applied to separate solutes as small as 1 nm [17][18][19][20] and as large as 0.1 mm [21,22].…”

Multimodal AGuIX® Nanoparticles: Size Characterization by HF5 and Optimization of the Radiolabeling with Various SPECT/PET/Theranostic Tracers

“…First, we developed a HF5method in aqueous media for the size-fractionation of AguiX NPs. These NPs were detected with satisfactory sensitivity, the method has been shown to be robust and reproducible [30].…”