Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

Isolation and Characterization of Exosomes from Ocular Samples.

Exosomes are microsize vesicles secreted by nearly all cells to the extracellular space. The vesicles transport cell signaling and communicate with other cells. Ultracentrifugation is the standard method to isolate exosomes from culture media or body fluid. Without ultracentrifuge, exosomes can be precipitated by polyethylene glycol or separated by size exclusion chromatography. After isolation, nanoparticle tracking analysis can help to estimate the size and concentration of exosome samples. Transmission electron microscopy can directly show the size and morphology of exosomes. Moreover, the sample should be characterized by the expression of several exosome biomarker proteins. Exosomal contents such as proteins and miRNAs could be profiled using appropriate technologies.

Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography.

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV-based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell-free plasma or serum. Because available one-step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2-step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV- and tumour-biomarkers in pure FPLC-derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases-associated biomarkers in pure EVs, supporting the feasibility of using single-vesicle analysis for liquid biopsy.

The emerging application of LC-based metallomics techniques to unravel the bioinorganic chemistry of toxic metal(loid)s.

The on-going anthropogenic emission of toxic metal(loid) species into the environment contaminates the food supply and drinking water resources in various parts of the world. Given that inorganic pollutants cannot be degraded, their increased influx into the bloodstream of babies, children and pregnant women is inevitable. Since the ramifications of the ensuing environmental exposure on human health remain poorly defined, fundamentally new insight into their bioinorganic chemistry in organisms is urgently needed. Based on the flow of dietary constituents through organisms, the interaction of toxic metal(loid) species with biomolecules in the bloodstream deserve particular attention as they play an integral role in the mechanisms of their chronic toxicity. Gaining insight into these bioinorganic processes is hampered by the biological complexity of plasma/red blood cells and the low concentrations of the metal(loid) species of interest, but can be overcome by employing LC techniques hyphenated to atomic spectroscopic detectors (i.e. metallomics techniques). This perspective aims to highlight the potential of unconventional hyphenated separation modes to advance our understanding of the bioinorganic chemistry of toxic metal(loid) species in the bloodstream-organ system. Four examples are illustrated. The application of anion-exchange (AEX) and size-exclusion chromatography (SEC) provided new insight into the blood-based bioinorganic mechanisms that direct Cd2+ and MeHg+ to target organs. AEX chromatography also allowed to observe the formation of complexes between Hg2+ and MeHg+ with L-cysteine at pH 7.4, that are implicated in their organ uptake. Lastly, the application of reversed phase (RP) chromatography revealed a possible cytosolic mechanism by which N-acetyl-L-cysteine binds to MeHg+ in the presence of cytosolic glutathione (GSH). New insight into other bioinorganic processes may advance the regulatory framework to better protect public health.

Measurement of α-synuclein as protein cargo in plasma extracellular vesicles.

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs in plasma. However, measuring cargo proteins in EVs has been challenging because EVs are present at low levels, and EV isolation methods are imperfect at separating EVs from free proteins. Thus, knowing whether a protein measured after EV isolation is truly inside EVs is difficult. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size-exclusion chromatography protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital enzyme-linked immunoassays (ELISAs) for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze α-synuclein and confirmed that a small fraction of the total plasma α-synuclein is inside EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated at the Ser129 residue). We found enrichment in the phosphorylated α-synuclein to total α-synuclein ratio inside EVs relative to outside EVs. Finally, we applied the methods we developed to measure total and phosphorylated α-synuclein inside EVs from Parkinson's disease and Lewy body dementia patient samples. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

Scaling laws for nanoparticles - Online shape heterogeneity analysis by size exclusion chromatography coupled with multi-angle light scattering.

Nanoparticle-based drug delivery systems are rising technologies to access challenging therapeutic targets. Following commercial success of lipid-based nanoparticles (LBNP), accruing understandings of nanoparticle structures and critical quality attributes through advanced analytics are beneficial to future clinical development and generalization of this delivery platform. The morphological attributes of nanoparticles, such as shape, can affect uptake, cell-interaction, drug release, circulation, and flow. Gaining an understanding of these structure-activity relationships in early-stage formulation development is important because mix morphologies can affect quality and potency but often exist before process control strategies are fully implemented. In this study, we used shape heterogeneous nanoparticle mixtures, containing various populations of liposomes and lipodisks, as a model system and developed an online semi-quantitative method to characterize the nanoparticle shape heterogeneity by size exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS). The liposomes and lipodisks were separated in SEC when their sizes were ∼3 fold different. When the particles of different shapes were in similar sizes, size-based separation was not always feasible. Instead, light scattering data distinguished liposomes and lipodisks by the scaling law linking radius of gyration and molecular weight of the nanoparticles, enabling morphological identification. A semi-quantitative model was built based on the exponential correlation between the scaling law exponents and the ratios of liposomes and lipodisks. The model was applied to test 6 random formulations made with different compositions and manufacturing processes, and the predicted liposome percentage for 5 formulations was within 25 % absolute difference from the percentage determined by cryogenic electron microscopy (cryo-EM). We envision this method being routinely used to characterize liposome and lipodisk shape heterogeneity during formulation screening as well as on stability studies. Potentially, the method can be converted to in-process control method and extended to other categories of nanoparticles beyond liposomes.

From Microsize Chromatographic Manufacturing for Fast Desalting to Its Characterization.

Microfluidic devices are becoming increasingly popular in protein analysis due to their ability to reduce sample and buffer volumes. However, there is a research gap concerning the coupling of this technology with ion mobility and mass spectrometry (IM-MS). This study aims to fill this void by introducing the manufacture and the characterization of a microsize exclusion chromatography (µSEC) module for fast desalting and its integration into microfluidics, along with its coupling to electrospray ionization and ion mobility mass spectrometry (ESI-IM-MS). To assess the feasibility of this approach, the desalting of α-synuclein (αS) was investigated using Bio Spin P6 gel as a stationary phase in the manufacture of a microfluidic device. αS detection by MS gives insight into the sample purity, while IM combined with MS provides information about protein structure. IM allowed both the recording of qualitative and quantitative information. The qualitative data provided a map of the conformers in equilibrium, while the calculation of the respective abundances (quantitative profile) of the conformers afforded the opportunity to describe the dynamics of the system. Our experiments, serving as proof-of-concept, demonstrate αS desalting, exchange buffer efficiency, and reduced solvent usage, without compromising the protein's structure.

A Novel Size Exclusion Chromatography Method for the Analysis of Monoclonal Antibodies and Antibody-drug Conjugates by Using Sodium Iodide in the Mobile Phase.

PURPOSES: Size exclusion chromatography (SEC) is widely used to characterize molecular size variants of antibody drugs. However, SEC analysis is hindered by secondary interactions (or nonspecific interactions) between proteins and stationary phase packing, which result in poor column efficiency. Previous studies have reported that chaotropic salt can inhibit these interactions, but the corresponding applications of this aspect are relatively rare. Therefore, this study introduces a novel approach using sodium iodide (NaI) as a mobile-phase component in SEC and investigates the influence of the mobile-phase composition on secondary interactions. METHODS: SEC analysis was performed on one antibody-drug conjugate and four monoclonal antibodies (mAbs) using three different mobile-phase systems (i.e., sodium chloride/L-arginine hydrochloride/NaI mobile phases system) to compare the column efficiency. Subsequently, mAb-1 was used as a model to investigate the effects of these factors on secondary interactions by adjusting the ionic strength (salt concentration) and pH of the NaI mobile-phase system. RESULTS: NaI exhibits superior column efficiency performance in the SEC analysis of most products. The ionic strength will affect nonideal electrostatic and hydrophobic interaction. An appropriate ionic strength can inhibit electrostatic interactions, while an excessive ionic strength increases hydrophobic interactions. pH primarily influences electrostatic interactions. Determining the appropriate pH necessitates consideration of the isoelectric point of the protein and the pH tolerance of the column. CONCLUSIONS: In SEC analysis, using NaI as the salt component in the mobile phase reduces secondary interactions and improves column efficiency. This approach is advantageous for samples with intense secondary interactions and is a suitable alternative.

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-derived Extracellular Vesicle-based Cancer Biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.

Enhancing monoclonal antibody stability during protein a chromatography using 2-methyl imidazolium dihydrogen phosphate.

This study investigates the impact of 2-methyl imidazolium dihydrogen phosphate (2-MIDHP) on monoclonal antibody (mAb) aggregation during the Protein A purification stage, at a low pH (pH 3.0), and the viral inactivation phase. Size-exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) were used to assess the mAb aggregation. Additionally, the influence of 2-MIDHP on mAb recovery, host cell protein (HCP) clearance, and Protein A leaching was investigated. Thermal stability of mAb, eluted in buffers containing 5 % to 25 % 2-MIDHP was analysed, using differential scanning calorimetry (DSC). Structural insights were obtained via circular dichroism (CD) and fluorescence spectroscopy. Our findings indicated that 2-MIDHP exerted a concentration-dependent protective effect against mAb aggregation, at the pH of 3.0. As the concentration of 2-MIDHP was increased from 0 % to 25 %, the aggregation was significantly reduced from 3.8 ± 0.01 % to 0.56 ± 0.002 %, as analysed by SE-HPLC. Addition of 2-MIDHP did not significantly impact the mAb recovery, HCP clearance, or Protein A leaching. DSC data supported these results, with higher 2-MIDHP concentrations leading to increased melting temperatures of mAb. CD and fluorescence spectroscopy revealed no significant changes in the secondary structure or aromatic residue environment in 2-MIDHP-treated samples, despite the observed reduction in aggregation. The results suggested that 2-MIDHP mitigated mAb aggregation during Protein A purification, possibly by stabilizing the protein structure under acidic stress conditions. These findings offer valuable insights for improving the robustness of mAb purification processes, enhancing product quality and yield.

Structural elucidation of Sulodexide with multidimensional chromatography and online in-source acid-induced dissociation mass spectrometry.

Sulodexide, a heparinoid medicine, is wildly used in clinic for prophylaxis and treatment of thromboembolic diseases and diabetic nephropathy. Despite its widespread use, the structure of Sulodexide remains poorly understood. It consists of various polysaccharides characterized by differing sugar compositions, linkages, and sulfonation patterns, yet they share common features such as strong hydrophilicity, high native charges, and considerable polydispersity, posing significant challenges for conventional chromatographic and online mass spectrometry (MS) characterization. In this work, a novel analytical method combining multiple-heart cut 2D-LC and in-source acid-induced dissociation (inAID) MS was developed. Three polysaccharides in Sulodexide were separated by high efficient strong-anion-exchange chromatography, followed by desalting with the second dimensional size-exclusion chromatography before MS. A novel MS strategy employing inAID technique was utilized for online analysis, leading to the initial identification of Sulodexide polysaccharide components. The results were validated through disaccharide composition analysis of those three polysaccharide components after offline preparation. This advanced strategy, merging various techniques, enable a comprehensive structural elucidation of such complex drugs and provides a viable tool for potential routine analysis of complex biomolecules.

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography.

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 - 78.2 °C for 10-100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1-3 °C, and denaturation power increases in the order MeOH

Capillary mediated vitrification is a novel technique that enables storage of antibody critical reagents at ambient temperature: Impact on binding, structure, and laboratory sustainability.

Antibodies and antibody conjugates are essential components of life science research, but their inherent instability necessitates cold storage or lyophilization, posing logistical and sustainability challenges. Capillary-mediated vitrification has shown promise as a tool for improving biomolecule stability. In this study, we assess the feasibility of shipping and storing CMV-stabilized antibody reagents at ambient temperature using a purified rabbit polyclonal as a model system. The conditions tested included a simulated temperature excursion, ambient shipping, and storage for approximately two months at room-temperature. Antibody function was measured by both ELISA and Octet bio-layer interferometry kinetic measurements. Yield, aggregation, and thermal stability were assessed by UV/VIS, Size Exclusion Chromatography (SEC), thermal melting, and thermal aggregation studies. Results indicate >97 % protein yield and no impact on the binding activity. No evidence of aggregation or oligomer formation was detected. Addition of the vitrification buffer to the sample matrix resulted in an increase in the aggregation on-set temperature, indicating enhanced thermostability. A slight shift in both the SEC retention time for the main peak and a difference in aggregation behavior at high temperatures were noted post-vitrification. We hypothesize that these differences are related to the interaction of the protein with the saccharide component of the vitrification matrix and the stabilization mechanism of sugars. The cumulative data supports the use of Capillary Mediated Vitrification as a viable alternative to frozen reagent storage, with the potential to significantly impact reagent stability, assay performance, laboratory operations, and sustainability initiatives.

Size-exclusion LC-UV/HRMS based method for the analysis of aggregates in synthetic GLP-1 analog liraglutide and evaluation of excipient impact on aggregation.

Peptide aggregation is one of the key challenges associated with the development of therapeutic peptides. Peptide and protein aggregates are considered as one of the most important critical quality attributes (CQA). Therapeutic liraglutide (LGT) is proteinaceous in nature, and aggregation can be triggered by various environmental stress condition. Therefore, it is essential to separate and identify aggregation states of such drugs. In this study, we have established size exclusion chromatography-liquid chromatography-ultraviolet/high resolution mass spectrometry (SEC-LC-UV/HRMS) method to separate and identify the stress induced LGT aggregates. LGT samples were subjected to photolytic, thermal, freeze thaw and shaking stress conditions. Additionally, LGT solution was incubated with surfactant and excipient that are commonly used in peptide formulation, to evaluate their impact on aggregation level and physicochemical stability over time. The developed SEC method was also validated for specificity, accuracy, precision and linearity. The results of this study will be useful for investigators to monitor LGT aggregates during product development.

Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Iron-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius): two-dimensional chromatographic separation with mass spectrometry detection.

Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-ß subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.

Protocol for the isolation and proteomic analysis of pathological tau-seeds.

The aggregation and spreading of "tau-seeds" are key for the development and progression of tauopathies, including Alzheimer's disease. Here we describe the steps to isolate and analyze biochemically active tau-seeds from human, mouse, and cell origin. We detail the procedure to isolate soluble tau-seeds by size exclusion chromatography and seeding assay. The isolated tau-seed can be further analyzed to determine the interactome by mass spectrometry. This workflow identifies protein-protein interactors of tau-seeds, providing a useful tool for finding new therapeutic targets. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.

Rapid determination of toxic and essential metal binding proteins in biological samples by size exclusion chromatography-inductively coupled plasma tandem mass spectrometry.

Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020-2.5 µg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.

Size exclusion chromatography and asymmetrical flow field-flow fractionation for structural characterization of polysaccharides: A comparative review.

Natural polysaccharides exhibit a wide range of biological activities, which are closely related to their structural characteristics, including their molecular weight distribution, size, monosaccharide composition, glycosidic bond types and spatial conformation, etc. Size exclusion chromatography (SEC) and asymmetrical flow field-flow fractionation (AF4), as two potent separation techniques, both harbor potential for continuous development and enhancement. This manuscript reviewed the fundamental principles and separation applications of SEC and AF4. The structural information and spatial conformation of polysaccharides can be obtained using SEC or AF4 coupled with multiple detectors. In addition, this manuscript elaborates in detail on the shear degradation of samples such as polysaccharides separated by SEC. In addition, the abnormal elution that occurs during the application of the two methods is also discussed. Both SEC and AF4 possess considerable potential for ongoing development and refinement, thereby offering increased possibilities and opportunities for polysaccharide separation and characterization.