Isolation of Extracellular Vesicles (EVs) Using Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC).

Extracellular vesicles (EVs), are membrane-bound nanoparticles of biological origin. These signature molecules of health and disease have raised remarkable attention of the biomedical arena due to its potential diagnostic and therapeutic applicability. Among the many different techniques available for EV isolation, size-exclusion chromatography (SEC) is widely accepted.In this chapter, we present a protocol of size-exclusion high-performance liquid chromatography (SE-HPLC) as a method of EV isolation. This method can be adapted as a low cost but a reliable and scalable method of EV isolation in those laboratories having access to the HPLC systems.

Isolation of Extracellular Vesicles (EVs) Using Benchtop Size Exclusion Chromatography (SEC) Columns.

A diverse group of lipid bilayer enclosed nanoparticles, referred to as extracellular vesicles (EVs), are released by all eukaryotic and prokaryotic cells into the extracellular space. The population of EVs being heterogeneous poses a challenge in their efficient separation. Several methods have been employed for EV isolation. However, there is no single conventional method that could recover a high amount of EVs while retaining their purity and functionality. The merging of differential centrifugation with size exclusion chromatography (SEC) is one of the best practices for EV isolation/purification as it recovers a sufficient amount of EVs while retaining their functionality. Here, we describe a method of purification of EVs from bovine follicular fluid samples using benchtop SEC columns. In conclusion, the EV purification method should be chosen based on the downstream applications, as every method poses its own limitations.

Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

Theoretical performance of multiple size-exclusion chromatography columns connected in series.

The fundamental relationships are derived for the retention, peak width, and peak capacity of non-retained polymers eluting from multiple standard size-exclusion chromatography (SEC) columns connected in series. The standard SEC columns may have different dimensions and are packed with particles having distinct average particle diameters (APDs) and average mesopore sizes (AMSs). The performances (peak capacity, local resolution power, and sensitivity) of three standard SEC columns connected in series (called a tri-SEC column) packed with bridged-ethylene-hybrid (BEH) fully porous particles (FPPs) having three different APDs (1.7, 2.5, and 3.5 µm) and AMSs (200, 450, and 900 Å, respectively) are calculated as a function of the applied flow rate and size of polystyrene standards. Irrespective of the APD and AMS, the present investigation assumes isomorphological materials relative to the mesopore space of the three different BEH particles. The advantage of a 15 cm long tri-SEC column over a single reference SEC column (APD=3.5 µm, AMS=900 Å), which generates the same back pressure and separation window as those of the tri-SEC column, is expected at flow rates larger than the optimum flow rate generating the maximum peak capacity. The calculations predict a significant relative increase of the peak capacity (from +25% to +85%), resolution of small molecules (from +75% to +225%), and of the detection limit of intermediate size (from +15% to +70%) and largest polymers (from +25 to +110%). This is explained by 1) the exclusion of the largest polymers from the internal volume of the particles having the smallest mesopores (restricted access media) and 2) the minimum dispersion along the columns packed with the smallest particle sizes in the tri-SEC column. The main benefit of multi-SEC columns is to easily adjust the desired pore size distribution by properly selecting the lengths of each individual SEC column. The user can then control the pore size distribution for any specific separation problem. A potential application is theoretically demonstrated for the fast purification of monoclonal antibodies from metabolites, host cell proteins, aggregated forms, and from virus-like particles.

Versatile, Sensitive, and Robust Native LC-MS Platform for Intact Mass Analysis of Protein Drugs.

Over the past several years, hyphenation of native (nondenaturing) liquid chromatography (nLC) methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) with mass spectrometry (MS) have become increasingly popular to study the size, charge, and structural heterogeneity of protein drug products. Despite the availability of a wide variety of nLC-MS methods, an integrated platform that can accommodate different applications is still lacking. In this study, we described the development of a versatile, sensitive, and robust nLC-MS platform that can support various nLC-MS applications. In particular, the developed platform can tolerate a wide range of LC flow rates and high salt concentrations, which are critical for accommodating different nLC methods. In addition, a dopant-modified desolvation gas can be readily applied on this platform to achieve online charge-reduction native MS, which improves the characterization of both heterogeneous and labile biomolecules. Finally, we demonstrated that this nLC-MS platform is highly sensitive and robust and can be routinely applied in protein drug characterization.

Combining online size exclusion chromatography and electrospray ionization mass spectrometry to characterize plant polysaccharides.

Characterizing polysaccharides with large molecular weights and isomeric heterogeneity with mass spectrometry (MS) is generally difficult. In this work, we demonstrate how coupling size exclusion chromatography (SEC) and high-resolution MS with source-induced dissociation (SID) can be used for the separation and direct structural evaluation of intact polysaccharides. The analytical method was successfully developed using dextran standards up to 3755 kDa. This method was used to separate naturally occurring plant polysaccharides based on size, after which numerous polysaccharide fragments were identified from the resulting MS spectra. The results provided strong evidence for structural diversity, complexity, and heterogeneity among polysaccharides. MS showed superior sensitivity and reliability for the polysaccharides in eluted fractions when compared to a refractive index detector. Putative compositions for the fragments were proposed based on exact mass values. The work demonstrated that SEC-SID-MS is a feasible alternative for obtaining valuable structural information from the analysis of intact polysaccharides.

Comprehensive online multicolumn two-dimensional liquid chromatography-diode array detection-mass spectrometry workflow as a framework for chromatographic screening and analysis of new drug substances.

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-µm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-µm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.

Isolation of Extracellular Vesicles from Leishmania spp.

Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.

Structural Analysis of RNA by Small-Angle X-ray Scattering.

Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY).

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Enhancing online protein isolation as intact species from soy flour samples by actively modulated two-dimensional liquid chromatography (2D-LC).

In this study, an enhanced fully automated approach is described for the protein isolation from soy flour samples by two-dimensional liquid chromatography with active modulation interface. The use of two multi-port switching valves is proposed to on-line connect the first to the second dimension column, thus overcoming the problems associated with the re-mixing effects and incompatibility of eluent composition and pH. A 5-cm long C4 analytical column installed in the interface device allows to focus the proteins coming from the first column (size exclusion chromatography), before their selective elution in the second column (reversed-phase). A trap washing step was included in the total workflow, as a desalting step to remove buffer residues from the eluent of the first column and to enhance the chromatographic performances of the second column. The experimental conditions were optimized by analyses of mixed standard solutions of bovine serum albumin, glucose oxidase, immunoglobulin A, thyroglobulin and myoglobin. Then, the optimized 2D-LC method was applied to the protein analysis in extracts of soy flour, known worldwide as one of the major food allergen sources, with the final aim to recovery sufficient protein amounts for the molecular characterization and the assessment of the pattern of allergenic components.

Biochemical and biophysical characterization of a prokaryotic Mg2+ ion channel: Implications for cost-effective purification of membrane proteins.

Although magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely used nonionic detergent, n-dodecyl-ß-d-maltopyranoside (DDM). However, DDM is an expensive detergent and alternative methods to produce high-quality proteins in stable and functional form will be practically advantageous to carry out structural studies in a cost-effective manner. In this work, we have utilized 'dual-detergent strategy' to successfully purify MgtE channel in a stable and functional form by employing relatively inexpensive detergents (Triton X-100 and Anzergent 3-14) for membrane solubilization and subsequently changed to DDM during purification. Our results show that Triton X-100 and Anzergent 3-14 extract MgtE well and the quality of purified protein is comparable to DDM-extracted MgtE. Interestingly, addition of high concentration of salt and glycerol during solubilization does not significantly affect the quantity and quality of MgtE. Importantly, limited proteolysis assay, circular dichroism spectroscopy and ensemble tryptophan fluorescence strongly support the use of Triton X-100, in particular, as an inexpensive, alternative detergent for the purification of MgtE without compromising the structural integrity of the channel and Mg2+-induced gating-related conformational dynamics. Overall, these results are relevant for the cost-effective purification of stable and functional membrane proteins in general, and magnesium channels, in particular.

Comprehensive two-dimensional liquid chromatography for the characterization of acrylate-modified hyaluronic acid.

Hyaluronic acid and its acrylate derivatives are important intermediates for various pharmaceutical, biomedical, and cosmetic applications due to their biocompatibility and viscoelasticity properties. However, these polymers are inherently difficult to characterize due to their significant heterogeneity regarding molar mass and chemical composition (degree of substitution, DS). The present study describes the development of a comprehensive online two-dimensional liquid chromatography (2D-LC) approach to characterize hyaluronic acid and its acrylate derivatives (DS ranging from 0.4 to 3.1) in terms of molar mass and degree of substitution. In the first dimension of the 2D-LC method, separation according to chemical composition/DS was achieved by using a stepwise solvent gradient and a reversed phase C8 column. Fractions from the first dimension were automatically transferred to the second dimension comprising size exclusion chromatographic separation of the fractions according to molar mass. It was found that the hyaluronic acid derivatives were broadly distributed with regard to both chemical composition and molar mass. Fractions with different degrees of substitution were identified, and their molar mass distributions were determined. The study proved that comprehensive 2D-LC is a powerful approach to reveal the complex nature of hyaluronic acid and its derivatives. Graphical abstract.

Isolation and Purification of a Neuroprotective Phlorotannin from the Marine Algae Ecklonia maxima by Size Exclusion and High-Speed Counter-Current Chromatography.

Phlorotannins are polyphenolic metabolites of marine brown algae that have been shown to possess health-beneficial biological activities. An efficient approach using a combination of high-speed counter-current chromatography (HSCCC) and size exclusion chromatography with a Sephadex LH-20 has been successfully developed for the isolation and purification of a neuroprotective phlorotannin, eckmaxol, from leaves of the marine brown algae, Ecklonia maxima. The phlorotannin of interest, eckmaxol, was isolated with purity >95% by HSCCC using an optimized solvent system composed of n-hexane-ethyl acetate-methanol-water (2:8:3:7, v/v/v/v) after Sephadex LH-20 size exclusion chromatography. This compound was successfully purified in the quantity of 5.2 mg from 0.3 kg of the E. maxima crude organic extract. The structure of eckmaxol was identified and assigned by NMR spectroscopic and mass spectrometric analyses. The purification method developed for eckmaxol will facilitate the further investigation and development of this neuroprotective agent as a drug lead or pharmacological probe. Furthermore, it is suggested that the combination of HSCCC and size exclusion chromatography could be more widely applied for the isolation and purification of phlorotannins from marine algae.

Serum Exosome Isolation by Size-Exclusion Chromatography for the Discovery and Validation of Preeclampsia-Associated Biomarkers.

Exosomes are extracellular nanovesicles of complex and heterogeneous composition that are released in biofluids such as blood. The interest in the characterization of exosomal biochemistry has increased over the last few years as they convey cellular proteins, lipids, and RNA that might reflect the biological or pathological condition of the source cell. In particular, association of changes of exosome proteins with specific pathogenic processes arises as a promising method to identify disease biomarkers as for the pregnancy-related preeclampsia. However, the overlapping physicochemical and structural characteristics of different types of extracellular vesicles have hindered the consolidation of universally accepted and standardized purification or enrichment protocols. Thus, it has been recently demonstrated that the exosomal protein profile resulting from in-depth proteomics analyses is highly dependent on the preparation protocol used, which will determine the particle type specificity and the presence/absence of contaminating proteins.In this chapter, an isolation method of serum exosomes based on size-exclusion chromatography (SEC) using qEV columns (Izon) is described. We show that this method is fast and reliable, as the population of exosomes isolated is homogeneous in terms of size, morphology, and protein composition. This exosome enrichment method is compatible with downstream qualitative and quantitative proteomic analysis of the samples.

A Top-Down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Identification and Regulation of Multimeric Protein Complexes in Autophagy via SILAC-Based Mass Spectrometry Approaches.

Mass spectrometry (MS)-based identification and characterization of protein complexes is becoming a prerequisite for in-depth biochemical analyses of intracellular processes. Here, we describe two state-of-the-art MS-based approaches to characterize protein-protein interactions and multi-protein complexes involved in autophagy in mammalian cells. The combination of affinity purification (AP)-MS, which identifies binary protein-protein interactions, with size-exclusion chromatography (SEC)-protein correlation profiling (PCP), which helps monitor protein complex assemblies, is a powerful tool to acquire a full overview of the interlinkage and regulation of novel multi-protein complexes that might play a role in autophagy.

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system.

The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition the amount of purified protein is usually in the µg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. In addition, a one-step purification procedure is often not sufficient to obtain pure protein preparations. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. The first chromatographic step is the capture step followed by a desalting step. The final purification was done using size exclusion chromatography. This set-up reduces the overall-time needed for protein production, needs minimal operator invention, is easy to handle and thus increases the throughput.

Concept of sample-specific correction of immunoassay results for precise and accurate IgG quantification in horse plasma.

The hyperimmune horse plasma (HHP), prepared through active immunisation of horses with an antigen of interest, is the most common starting material for antitoxin (animal antibody-based therapeutics) production. Precise IgG quantification in plasma is a prerequisite for accurate estimation of the purification process efficiency. Although immunoglobulins from HHP have been purified for over a century, there is still no in vitro method for precise and accurate determination of IgG content in HHP. For this reason, the purification process efficiency has been assessed by antibody activity measurements, mostly performed in vivo. Here we describe the development of a precise and accurate in vitro immunoassay for IgG quantification in HHP. We showed and highlighted that any difference in composition of IgG population between the standard and the sample, with respect to both IgG subclass distribution and antigen-specific IgG content, leads to inaccurate IgG quantification. We demonstrated that caprylic acid precipitation as the method for IgG isolation from horse plasma renders the composition of IgG population unchanged. This very efficient, fast, simple and inexpensive method was used to prepare internal, sample-specific reference IgG for each plasma sample, which was tested simultaneously to a respective plasma sample. Deviation of IgG quantity determined by ELISA for each sample-specific reference from its nominal value was used for correction of the results of respective plasma sample, which led to accurate and precise IgG quantification as shown by method validation. The here presented novel concept of sample-specific correction of immunoassay results could be widely applicable and easily introduced in different immunoassays for more accurate and precise plasma IgG quantification.

Assembly of PGAM5 into Multimeric Complexes Provides a Mechanism for Allosteric Regulation of Phosphatase Activity.

Phosphoglycerate mutase family member 5 (PGAM5) is a serine/threonine phosphatase that has been localized to both inner and outer mitochondrial membranes. PGAM5 has been suggested to regulate multiple aspects of mitochondrial dynamics, including fission/fusion and mitophagy, through phosphatase-dependent and phosphatase-independent mechanisms. Understanding how the phosphatase activity of PGAM5 is regulated will provide new insight into signaling mechanisms that link changes in cell physiology with mitochondrial function. In this chapter, we describe methods for obtaining both multimeric and dimeric complexes of PGAM5 and for characterizing their kinetic properties. The ability to purify different PGAM5 complexes and to characterize their kinetic properties will enable detailed biophysical studies of the quaternary structures of the various PGAM5-containing complexes. The phosphatase activity of different PGAM5 complexes varies over three orders of magnitude. We suggest that the ability to generate PGAM5 complexes that have a wide range of phosphatase activities will facilitate screens to identify small molecules that modulate the phosphatase activity of PGAM5.