A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

Hong, Paula; Koza, Stephan M.; Bouvier, Edouard S. P.

doi:10.1080/10826076.2012.743724

Cited by 459 publications

(317 citation statements)

References 117 publications

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“…The size (hydrodynamic molecular mass, m ) of heat‐induced soluble SPI aggregates and the m distribution were characterized with size exclusion HPLC. The method yielded reproducible results in agreement with literature reports (Hong and others ). Attempts were made to also use light scattering techniques, including multiangle laser light scattering (MALLS) and dynamic light scattering (Zetasizer), to measure the particle sizes.…”

Section: Resultssupporting

confidence: 90%

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

Guo

Xiong

Qin

et al. 2015

Journal of Food Science

102

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Suspensions (2% and 5%, w/v) of soy protein isolate (SPI) were heated at 80, 90, or 100 °C for different time periods to produce soluble aggregates of different molecular sizes to investigate the relationship between particle size and surface properties (emulsions and foams). Soluble aggregates generated in these model systems were characterized by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat treatment increased surface hydrophobicity, induced SPI aggregation via hydrophobic interaction and disulfide bonds, and formed soluble aggregates of different sizes. Heating of 5% SPI always promoted large-size aggregate (LA; >1000 kDa) formation irrespective of temperature, whereas the aggregate size distribution in 2% SPI was temperature dependent: the LA fraction progressively rose with temperature (80→90→100 °C), corresponding to the attenuation of medium-size aggregates (MA; 670 to 1000 kDa) initially abundant at 80 °C. Heated SPI with abundant LA (>50%) promoted foam stability. LA also exhibited excellent emulsifying activity and stabilized emulsions by promoting the formation of small oil droplets covered with a thick interfacial protein layer. However, despite a similar influence on emulsion stability, MA enhanced foaming capacity but were less capable of stabilizing emulsions than LA. The functionality variation between heated SPI samples is clearly related to the distribution of aggregates that differ in molecular size and surface activity. The findings may encourage further research to develop functional SPI aggregates for various commercial applications.

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Section: Resultssupporting

confidence: 90%

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

Guo

Xiong

Qin

et al. 2015

Journal of Food Science

102

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“…The analytical SEC, in conjunction with the native PAGE analysis, suggest that after a 2 h treatment with DTT and EDTA, SOD1 undergoes a conformational change that affects the hydrodynamic volume and the packing of the protein. It is well known that changes to the structure of molecules can make SEC data ambiguous (Hong et al, 2012), potentially making the interpretation of these data complicated. Due to this, native mass spectrometry analysis was performed on fractions eluted from analytical SEC assays.…”

Section: Resultsmentioning

confidence: 99%

“…Investigation of these possible unfolded SOD1 conformations has been difficult due to the heterogeneity of non-native protein structures. High resolution analyses, such as X-ray crystallography, are impeded by the existence of disordered protein regions that can assume multiple conformations (Banci et al, 2009), and typical separation techniques, such as size exclusion chromatography (SEC) do not provide sufficient resolution to separate different conformations for downstream analysis (Hong et al, 2012), although use of small-angle X-ray scattering coupled with SEC can provide valuable insight into the structural plasticity of proteins in solution by separating different conformers and oligomeric states (Wright et al, 2011, 2016; Furukawa et al, 2015). Nuclear magnetic resonance (NMR) spectroscopy has been used to great extent on disordered SOD1, monitoring unfolding, refolding, and even identifying a zinc-deficient form of SOD1 proposed as a precursor to the toxic species (Luchinat et al, 2014; Szpryngiel et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity

2016

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (<1 year to ~17 years death post-diagnosis) that are mutation dependent. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R.

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“…That is not the case of the proteins markers used, which are close to spheres. However, underestimation of molecular weight by SEC is conceivable as well [25]. …”

Section: Resultsmentioning

confidence: 99%

Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1

Chamizo-Ampudia

Galván

Fernández

et al. 2017

IJMS

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The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction of several N-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this protein in the green alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b5 (crCytb5-1) and a cytochrome b5 reductase (crCytb5-R) that form an electron transport chain from NADH to the substrates. Here, we have investigated NHC reduction by crARC, the interaction with its partners and the function of important conserved amino acids. Interactions among crARC, crCytb5-1 and crCytb5-R have been studied by size-exclusion chromatography. A protein complex between crARC, crCytb5-1 and crCytb5-R was identified. Twelve conserved crARC amino acids have been substituted by alanine by in vitro mutagenesis. We have determined that the amino acids D182, F210 and R276 are essential for NHC reduction activity, R276 is important and F210 is critical for the Mo Cofactor chelation. Finally, the crARC C-termini were shown to be involved in protein aggregation or oligomerization.

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A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

Cited by 459 publications

References 117 publications

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity

Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1

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