The Emerging Role of Exosome Nanoparticles in Regenerative Medicine
Harishkumar Madhyastha, Durgesh Nandini Chauhan in Nanopharmaceuticals in Regenerative Medicine, 2022
The EVs (especially the exosomes) are most commonly isolated by the ultracentrifugation method. This conventional technique is based on the size of the particle (commonly by >1,00,000g as speed and ~5 hours as time). The exosomes could also be precipitated using special reagents (e.g. polymeric additives). The basis for commercial exosome isolation kits is the ~30-min precipitation by a standard centrifuge (~10,000g). Another commercial exosome isolation method is the membrane filters (e.g. polyvinylidene difluoride (PVDF) or polycarbonate with pore size of 50–450 nm). The chromatography technique could also be employed for exosome isolation. The commercial size-exclusion chromatography columns are provided to induce the isolation of the exosomes within eluted fractions, which contain larger to smaller particles.
Experimental Strategies
Clive R. Bagshaw in Biomolecular Kinetics, 2017
Gel-permeation (size-exclusion) chromatography can also be adapted for quantitative analysis of binding. Care is required in using chromatography-based methods to quantify binding because some knowledge of the kinetics of the reaction relative to the separation time is required (cf. comments in the Electrophoresis section). In the Hummel and Dreyer method [330,331], the column is equilibrated with buffer containing the ligand at a fixed concentration. The macromolecule is then added to the column in a small volume. If the macromolecule binds to the ligand, then the latter will be carried ahead of the free ligand because it will be excluded from the column material. A peak in the elution profile of the ligand will be observed, followed by a trough of equal magnitude due to local depletion of the ligand. The area under the peak or trough provides a measure of the bound ligand. This method assumes that the ligand binds on a time scale shorter than the column separation time. For slowly equilibrating species, a kinetic method is better employed (see “spin columns,” Section 7.2.5).
Proteins in Cosmetics
E. Desmond Goddard, James V. Gruber in Principles of Polymer Science and Technology in Cosmetics and Personal Care, 1999
Size exclusion chromatography (also referred to as gel filtration and gel permeation chromatography) is the most practical and frequently employed technique to fractionate polypeptides in function of their dimension and provides the basis to estimate the average molecular weight of protein blends. Exclusion columns contain porous particles of a hydrophilic polymer with different pore diameters. When a polypeptide mixture is forced to pass through the column, its components diffuse into those pores that have a diameter greater than their effective diameters (the effective diameter will include any molecules of solvation). If a solute is of such a diameter that it cannot diffuse into any of the pores, it will be unretained and elute in the void volume of the column. Conversely, a polypeptide that is capable of diffusing completely into all of the pores will totally permeate the packing and require a much larger volume of solvent to achieve elution. The separation of molecules having different diameters is based on the extent of their diffusion through the pores and the different time needed to achieve their elution. An ideal size exclusion fractionation will occur when all solutes diffuse into part of the pores of the stationary phase. Size exclusion chromatography is a way of rapidly obtaining an estimate of the molecular weight of a sample, by comparison with authentic standards. To correlate the elution time of the polypeptides (which is a function of their size) with their molecular weight, the solutes and standards should have the same spatial conformation. For blends of molecules having homogeneous shape, over a considerable range, the elution volume is approximately a linear function of the logarithm of molecular weight. Practically, molecular masses of polypeptides are only approximately determinable as their hydrodynamic
Unraveling the complexity of the extracellular vesicle landscape with advanced proteomics
Published in Expert Review of Proteomics, 2022
Julia Morales-Sanfrutos, Javier Munoz
EVs can be separated from soluble proteins and protein aggregates by density gradient ultracentrifugation (DG), since vesicles have lower densities (1.13–1.19 g/mL) than proteins (1.35–1.41 g/mL). In this strategy, a continuous or discontinuous pre-constructed density gradient, which increases in density from top to bottom, is used. Different mediums are available, being sucrose and iodixanol (Optiprep) most commonly used. Upon ultracentrifugation for several hours, sEVs (including exosomes) migrate to the point where their density is the same as the medium density. DG is very effective in separating sEVs from proteins and non-membranous contaminations, avoiding vesicle aggregation, and can even separate EVs subtypes. However, these benefits are accompanied by increased costs, time and workload. In addition, this strategy requires an extra step, such as ultracentrifugation or ultrafiltration, to remove the density medium prior to MS analysis. In combination with dUC, DG was one of the first attempts to address the heterogeneity of EVs [18].(3) Size-exclusion chromatography
Small extracellular vesicles (sEVs): discovery, functions, applications, detection methods and various engineered forms
Published in Expert Opinion on Biological Therapy, 2021
Manica Negahdaripour, Hajar Owji, Sedigheh Eskandari, Mozhdeh Zamani, Bahareh Vakili, Navid Nezafat
Polymer-based precipitation methods are alternative strategies used for sEV isolation, which relies on the hydrophobic interactions between the exosomal membrane components and precipitation reagents [124,132]. Until now, several companies have introduced PEG-based separation kits to rapidly separate sEVs [133]. Despite offering many benefits, these commercial separation kits are expensive and exhibit a low purity rate due to the co-precipitation of other protein contaminations. Another technique that has gained popularity in recent years is size exclusion chromatography (SEC). Compared with PEG-based precipitation and PRotein Organic Solvent PRecipitation (PROSPR)-based isolation methods, SEC potently removed protein contaminations during extracellular vesicle (EV) isolation procedure. Moreover, the EV-markers, such as CD9, CD63, CD81, LGALS3BP, and CD5L, were only detected by SEC [134].
The role of mass spectrometry in the characterization of biologic protein products
Published in Expert Review of Proteomics, 2018
Deepali Rathore, Anneliese Faustino, John Schiel, Eric Pang, Michael Boyne, Sarah Rogstad
HPLC and UHPLC are also often used as standalone techniques to study structural heterogeneity of intact proteins. For example, strong-cation exchange can be used to resolve charge state variants and to identify charge-related degradation products (e.g. deamidation) [37,38]. Similarly, size exclusion chromatography and hydrophobic interaction chromatography are often used to analyze size- and hydrophobic-variants, respectively [38]. Off-line analysis is especially useful when using off-site mass spectrometers, when analysis of only a portion of chromatogram is required, when direct coupling of these techniques with MS is limited due to mobile phase incompatibility with MS and for multiplexing slower separations to rapid MS analysis. Capillary electrophoresis (CE) is another separation technique used for the characterization of biotechnology products that is rapid, reproducible, and highly sensitive [38,39]. When operating in capillary isoelectric focusing or capillary zone electrophoresis modes, CE can provide information on the charge variants [40]. CE coupled with MS (CE-MS) has been used to separate and identify intact proteins [41], PTMs including signal peptide removal, N-terminal methionine excision, and acetylation [42].