Range Of Protein Concentrations Research Articles

Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, De /D0, remains constant and has a value of 0.20. The BiSAb's structure is comprised of scFv domains that are genetically fused to a framework IgG through flexible peptide linkers which results in conformational flexibility leading to multiple binding forms with varying affinity for the adsorbent surface. As a result, adsorption of the BiSAbs shows complex patterns of total bound protein within the particles. These BiSAb adsorption patterns are influenced by buffer ionic strength, pH, and temperature in unique ways. Sharper intraparticle profiles are observed for conditions where the binding strength is greater (lower buffer concentration and/or pH) or when the protein is chemically crosslinked to restrict configurational flexibility. Temperature affects the BiSAb pore diffusivity as well as the interconversion kinetics. While the effects of temperature on BiSAb transport are also described by a constant De /D0 = 0.15, the temperature also affects the rate of interconversion between binding forms leading to faster equilibration at higher temperatures. A phenomenological model indicates that the interplay of pore diffusion and adsorption with the kinetically limited interconversion between binding forms is responsible for the experimental trends.

Protein isolates from Cajanus cajan L. as surfactant for o:w emulsions: pH and ionic strength influence on protein structure and emulsion stability

This work has focused on the study of the influence of pH (2.1, 3.9, 6.3, 8.3) and ionic strength (μ) (0.10 and 0.54) on the formation and stability of oil in water (o:w) emulsions using a protein isolate of Cajanus cajan (PPI8) as emulsifier. Also, the influence of protein concentration was evaluated. Interfacial tension was better at pH2.1 and pH8.3, and it was improved at pH6.3 with the increase in ionic strength. Most emulsions showed a monomodal droplet size distribution, except at pH8.3 (μ: 0.54) where a bimodal distribution was observed. The critical concentration range of proteins was between 1.5 and 2.0% (μ: 0.10) and 1.0–1.5% (μ: 0.54). At pH6.3, the surface average diameter was independent of ionic strength (0.5%, 1.0%, and 2.0%). At low ionic strength, stable emulsions (protein concentration ≥1.0%) were obtained (up to 7 days) at pH2.1, pH6.3, and pH8.3, despite the differences in flocculation index (FI) observed. FI of emulsions remained constant up to 7 days of storage, except at pH2.1 (1.0%) and pH8.3 (2.0%). The emulsion stability at high ionic strength was pH dependent, at pH3.9 was improved even at low protein concentration due to the increase in protein solubility and apparent viscosity of emulsion. Because of their techno-functional properties, PPI8 could lead to the development of innovative products suitable for those consumers that demands for alternative plant-based protein sources.

Effects of preceding crops and nitrogen fertilizer on the productivity and quality of malting barley in tropical environment

The growing demand for malt has generated interest for improving productivity through sustainable means such as cropping sequences with malting barley along with optimum nitrogen (N) fertilization. Cropping sequence has many benefits for optimum yield and quality, but knowledge of rotational effects of preceding crops on malting barley is still limited. Thus, this study was conducted to determine the effects of legume and non-legume preceding crops, and N fertilization on productivity and quality of malting barley grown the following year in two locations in the southeastern highland of Ethiopia. The experiment was split plot design with six preceding crops (fababean, Ethiopian mustard, potato, linseed, wheat and malting barley) as main plots and four levels of N (0, 18, 36 and 54 kg N ha−1) for the succeeding crop as split plot treatments with 3 replications. Malting barley grown after fababean, Ethiopian mustard and potato exhibited 13–16, 14–34 and 14%, respectively grain yield increments compared to growing malting barley after malting barley. Similarly, application of 36–54 kg N ha−1 gave 4–29 and 3–19% grain yield increments compared to the control (no N) and previous recommendation (18 kg N ha−1), respectively with no detrimental effect on kernel protein concentrations. Seeding malting barley at a rate of 54 kg N ha−1 gave 250–915% increase in economic benefit. Use of break crops other than barley and increasing the rate of N application from 18 to 54 kg ha−1 have been recommended to boost malting barley yield without surpassing the acceptable range of kernel protein concentrations, reduce costs of production, increase profitability and improve soil fertility to enhance long-term sustainability of the cropping system.

Fast SARS-CoV-2 virus detection using disposable cartridge strips and a semiconductor-based biosensor platform.

Detection of the SARS-CoV-2 spike protein and inactivated virus was achieved using disposable and biofunctionalized functional strips, which can be connected externally to a reusable printed circuit board for signal amplification with an embedded metal-oxide-semiconductor field-effect transistor (MOSFET). A series of chemical reactions was performed to immobilize both a monoclonal antibody and a polyclonal antibody onto the Au-plated electrode used as the sensing surface. An important step in the biofunctionalization, namely, the formation of Au-plated clusters on the sensor strips, was verified by scanning electron microscopy, as well as electrical measurements, to confirm successful binding of thiol groups on this Au surface. The functionalized sensor was externally connected to the gate electrode of the MOSFET, and synchronous pulses were applied to both the sensing strip and the drain contact of the MOSFET. The resulting changes in the dynamics of drain waveforms were converted into analog voltages and digital readouts, which correlate with the concentration of proteins and virus present in the tested solution. A broad range of protein concentrations from 1 fg/ml to 10 μg/ml and virus concentrations from 100 to 2500 PFU/ml were detectable for the sensor functionalized with both antibodies. The results show the potential of this approach for the development of a portable, low-cost, and disposable cartridge sensor system for point-of-care detection of viral diseases.

β-Lactoglobulin Adsorption Layers at the Water/Air Surface: 5. Adsorption Isotherm and Equation of State Revisited, Impact of pH

The theoretical description of the adsorption of proteins at liquid/fluid interfaces suffers from the inapplicability of classical formalisms, which soundly calls for the development of more complicated adsorption models. A Frumkin-type thermodynamic 2-d solution model that accounts for nonidealities of interface enthalpy and entropy was proposed about two decades ago and has been continuously developed in the course of comparisons with experimental data. In a previous paper we investigated the adsorption of the globular protein β-lactoglobulin at the water/air interface and used such a model to analyze the experimental isotherms of the surface pressure, Π(c), and the frequency-, f-, dependent surface dilational viscoelasticity modulus, E(c)f, in a wide range of protein concentrations, c, and at pH 7. However, the best fit between theory and experiment proposed in that paper appeared incompatible with new data on the surface excess, Γ, obtained from direct measurements with neutron reflectometry. Therefore, in this work, the same model is simultaneously applied to a larger set of experimental dependences, e.g., Π(c), Γ(c), E(Π)f, etc., with E-values measured strictly in the linear viscoelasticity regime. Despite this ambitious complication, a best global fit was elaborated using a single set of parameter values, which well describes all experimental dependencies, thus corroborating the validity of the chosen thermodynamic model. Furthermore, we applied the model in the same manner to experimental results obtained at pH 3 and pH 5 in order to explain the well-pronounced effect of pH on the interfacial behavior of β-lactoglobulin. The results revealed that the propensity of β-lactoglobulin globules to unfold upon adsorption and stretch at the interface decreases in the order pH 3 > pH 7 > pH 5, i.e., with decreasing protein net charge. Finally, we discuss advantages and limitations in the current state of the model.

Line-FRAP, A Versatile Method to Measure Diffusion Rates In Vitro and In Vivo

The crowded cellular milieu affect molecular diffusion through hard (occluded space) and soft (weak, non-specific) interactions. Multiple methods have been developed to measure diffusion coefficients at physiological protein concentrations within cells, each with its limitations. Here, we show that Line-FRAP, combined with rigours data analysis, is able to determine diffusion coefficients in a variety of environments, from in vitro to in vivo. The use of Line mode greatly improves time resolution of FRAP data acquisition, from 20-100 Hz in the classical mode to 800 Hz in the line mode. This improves data analysis, as intensity and radius of the bleach at the first post-bleach frame is critical. We evaluated the method on different proteins labelled chemically or fused to YFP in a wide range of environments. The diffusion coefficients measured in HeLa and in E. coli were ~2.5-fold and 15-fold slower than in buffer, and were comparable to previously published data. Increasing the osmotic pressure on E. coli further decreases diffusion, to the point at which proteins virtually stop moving. The method presented here, which requires a confocal microscope equipped with dual scanners, can be applied to study a large range of molecules with different sizes, and provides robust results in a wide range of environments and protein concentrations for fast diffusing molecules.

Unbiased Detection of Cysteine Sulfenic Acid by 473 nm Photodissociation Mass Spectrometry: Toward Facile In Vivo Oxidative Status of Plasma Proteins.

Cysteine (Cys) is prone to diverse post-translational modifications in proteins, including oxidation into sulfenic acid (Cys-SOH) by reactive oxygen species generated under oxidative stress. Detection of low-concentration and metastable Cys-SOH within complex biological matrices is challenging due to the dynamic concentration range of proteins in the samples. Herein, visible laser-induced dissociation (LID) implemented in a mass spectrometer was used for streamlining the detection of Cys oxidized proteins owing to proper derivatization of Cys-SOH with a chromophore tag functionalized with a cyclohexanedione group. Once grafted, peptides undergo a high fragmentation yield under LID, leading concomitantly to informative backbone ions and to a chromophore reporter ion. Seventy-nine percent of the Cys-containing tryptic peptides derived from human serum albumin and serotransferrin tracked by parallel reaction monitoring (PRM) were detected as targets subjected to oxidation. These candidates as well as Cys-containing peptides predicted by in silico trypsin digestion of five other human plasma proteins were then tracked in real plasma samples to pinpoint the endogenous Cys-SOH subpopulation. Most of the targeted peptides were detected in all plasma samples by LID-PRM, with significant differences in their relative amounts. By eliminating the signal of interfering co-eluted compounds, LID-PRM surpasses conventional HCD (higher-energy collisional dissociation)-PRM in detecting grafted Cys-SOH-containing peptides and allows now to foresee clinical applications in large human cohorts.

Multiprotein E. coli SSB-ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions.

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.

Proteomic Analysis of Chr 18 Proteins Using 2D Fractionation.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of "missing proteins" (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5-10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.

BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy.

Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold.The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.

Selective enrichment of N-terminal proline peptides via hydrazide chemistry for proteomics analysis

A challenge for shotgun proteomics is the identification of low abundance proteins, which is always hampered owing to the extreme complexity of protein digests and highly dynamic concentration range of proteins. To reduce the complexity of the peptide mixture, we developed a novel method to selectively enrich N-terminal proline peptides via hydrazide chemistry. This method consisted of ortho-phthalaldehyde (OPA) blocking of primary amines in peptides, reductive glutaraldehydation of N-terminal proline and solid phase hydrazide chemistry enrichment of aldehyde-modified N-terminal proline peptide. After enrichment, the number of detected peptides containing N-terminal proline increased from 1304 to 4039 and the ratio of N-terminal proline peptides jumped from 4.4% to 93.7%, showing good enrichment specificity towards N-terminal proline peptides. Besides, the ratio of identified peptides to proteins was decreased from 7.8 (29751/3811) to 1.5 (4347/2821), indicating that sample complexity was drastically reduced through this method. As a result, this novel approach for enriching N-terminal proline peptides is effective in identification of low abundance protein owing to the reduction of sample complexity.

An Integrated Quantitative Proteomics Workflow for Cancer Biomarker Discovery and Validation in Plasma.

Blood plasma is one of the most widely used samples for cancer biomarker discovery research as well as clinical investigations for diagnostic and therapeutic purposes. However, the plasma proteome is extremely complex due to its wide dynamic range of protein concentrations and the presence of high-abundance proteins. Here we have described an optimized, integrated quantitative proteomics pipeline combining the label-free and multiplexed-labeling-based (iTRAQ and TMT) plasma proteome profiling methods for biomarker discovery, followed by the targeted approaches for validation of the identified potential marker proteins. In this workflow, the targeted quantitation of proteins is carried out by multiple-reaction monitoring (MRM) and parallel-reaction monitoring (PRM) mass spectrometry. Thus, our approach enables both unbiased screenings of biomarkers and their subsequent selective validation in human plasma. The overall procedure takes only ~2 days to complete, including the time for data acquisition (excluding database searching). This protocol is quick, flexible, and eliminates the need for a separate immunoassay-based validation workflow in blood cancer biomarker investigations. We anticipate that this plasma proteomics workflow will help to accelerate the cancer biomarker discovery program and provide a valuable resource to the cancer research community.

Insight of low-abundance proteins in rice leaves under Cd stress using combinatorial peptide ligand library technology.

Low-abundance proteins (LAPs) play a very important role in interaction, regulation, and metabolism of plant biological processes. A combinatorial peptide ligand library (CPLL) can solve the problem of high-abundance proteins (HAPs) masking LAPs and enlarging the dynamic range of protein concentrations perfectly and be considered as one of the most advanced approaches for plant proteomics research. In this paper, a proper CPLL method to rice leaf proteins was established for the first time and 1056 proteins were identified in rice leaf extracts, and 624 (59.1%) LAPs were newly detected after CPLL. Based on this technology, we detected the response of rice to Cd stress and analyzed the differential LAPs and the biological significance of misexpressed proteins before and after Cd stress by bioinformatics analysis. An important contribution has also been made to a better understanding of the complex mechanisms by which rice adapts to Cd stress. Graphical abstract.

Nano-scavengers for blood biomarker discovery in ovarian carcinoma

The development and implementation of biomarker-based screening tools for ovarian cancer require novel analytical platforms to enable the discovery of biomarker panels that will overcome the limitations associated with the clinically used CA-125.The systematic discovery of protein biomarkers directly from human plasma using proteomics remains extremely challenging, due to the wide concentration range of plasma proteins. Here, we describe the use of lipid-based nanoparticles (NPs) as an ‘omics’ enrichment tool to amplify cancer signals in the blood and to uncover disease specific signatures. We aimed to exploit the spontaneous interaction of clinically-used liposomes (Caelyx®) with plasma proteins, also known as’ protein corona’ formation, in order to facilitate the discovery of previously unreported differentially abundant molecules. Caelyx® liposomes were incubated with plasma samples obtained from advanced ovarian carcinoma patients and healthy donors and corona-coated liposomes were subsequently recovered. Comprehensive comparison between ‘healthy’ and ‘diseased’ corona samples by label-free proteomics resulted in the identification of multiple differentially abundant proteins. Moreover, immunoassay-based validation of selected proteins demonstrated the potential of nanoparticle-platform proposed to discover novel molecules with great diagnostic potential. This study proposes a nanoparticle-enabled workflow for plasma proteomic analysis in healthy and diseased states and paves the way for further work needed to discover and validate panels of novel biomarkers for disease diagnosis and monitoring.

Aptamer-Gold(III) Complex Nanoparticles: A New Way to Detect Cu, Zn SOD Glycoprotein.

Aptamers are small biomolecules composed of 20–100 nucleotides that recognize target molecules in three-dimensional structures. These natural targeting molecules have attracted interest in the biomedical field as biomarkers for cancer diagnostics. In this study, we investigated the interaction of a characteristic aptamer with its target protein, Cu, Zn superoxide dismutase (SOD 4), on a gold nanoparticle (AuNP) surface under experimental conditions. For this purpose, we applied two protocols to coat SOD 4 aptamer (APT) on the nanoparticle surface: carbodiimide chemistry (EDC/NHS) (Method ON) and a complexation methodology (Method IN). The nano-aptamer’s interactions with SOD 4 were detected by UV–vis absorption and Raman spectroscopy in a range of protein concentrations (from 1 μM to 50 nM). We believe that the interaction is heavily dependent on the nature of the biomarker (SOD 4) and also on the steric arrangement of the aptamer on the gold nanoparticle surface. The lowest detectable concentration (limit of detection, LOD) was about 2 nM for APT IN PEG-AuNPs and 8 nM for APT ON PEG-AuNPs. For the first time, we demonstrated a very sensitive detection of SOD 4 in the nanomolar concentration range with new ways of biosensor synthesis (APT IN and ON), providing a very strong tool to understand the effect of aptamer conformation to detect SOD 4.

C-terminal eYFP fusion impairs Escherichia coli MinE function.

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

Coarse-Grained Molecular Dynamics Simulations for Understanding the Impact of Short-Range Anisotropic Attractions on Structure and Viscosity of Concentrated Monoclonal Antibody Solutions.

Understanding protein-protein interactions in concentrated therapeutic monoclonal antibody (mAb) solutions is desirable for improved drug discovery, processing, and administration. Here, we deduce both the net protein charge and the magnitude and geometry of short-ranged, anisotropic attractions of a mAb across multiple concentrations and cosolute conditions by comparing structure factors S(q) obtained from small-angle X-ray scattering experiments with those from molecular dynamics (MD) simulations. The simulations, which utilize coarse-grained 12-bead models exhibiting a uniform van der Waals attraction, uniform electrostatic repulsion, and short-range attractions between specific beads, are versatile enough to fit S(q) of a wide range of protein concentrations and ionic strength with the same charge on each bead and a single anisotropic short-range attraction strength. Cluster size distributions (CSDs) obtained from best fit simulations reveal that the experimental structure is consistent with small reversible oligomers in even low viscosity systems and help quantify the impact of these clusters on viscosity. The ability to systematically use experimental S(q) data together with MD simulations to discriminate between different possible protein-protein interactions, as well as to predict viscosities from protein CSDs, is beneficial for designing mAbs and developing formulation strategies that avoid high viscosities and aggregation at high concentration.

β-Lactoglobulin Adsorption Layers at the Water/Air Surface: 3. Neutron Reflectometry Study on the Effect of pH.

Several characteristics of β-lactoglobulin (BLG) layers adsorbed at the air/water interface exhibit a strong pH dependence, but our knowledge on the underlying structure-property relations is still fragmental. Here, we therefore extend our recent studies by neutron reflectometry (NR) and provide a comprehensive overview through direct measurements of the surface excess Γ and the layers' molecular structure. This enables comparison with available literature data to draw general conclusions. The NR experiments were performed at various pH values and within a wide range of protein concentrations, CBLG. Adsorption kinetics measurements in air-contrast-matched-water and over a narrow Qz range enabled direct quantification of the dynamic surface excess Γ(t) and are found to be consistent with ellipsometry data. Near the isoelectric point, pI, the rates of adsorption and Γ are maximal but only at sufficiently high CBLG. NR data collected over a wider Qz range and in two aqueous isotopic contrasts revealed the structure of adsorbed BLG layers at a steady state close to equilibrium. Independent of the pH, BLG was found to form dense monolayers with average thicknesses of 1.1 nm, suggesting flattening of the BLG globules upon adsorption as compared with their bulk dimensions (≈3.5 nm). Near pI and at sufficiently high CBLG, a thick (≈5.5 nm) but looser secondary sublayer is additionally formed adjacent to the dense primary monolayer. The thickness of this sublayer can be interpreted in terms of disordered BLG dimers. The results obtained and notably the specific interfacial structuring of BLG near pI complement previous observations relating the impact of solution pH and CBLG on other interfacial characteristics such as surface pressure and surface dilational viscoelasticity modulus.

Blood plasma high abundant protein depletion unintentionally carries over 100 proteins

AbstractThere is a constant interest in blood‐based protein biomarkers, which can help to improve diagnosis and treatment outcomes of multifactorial human pathologies. In this regard, proteomic studies usually employ plasma immunoaffinity fractionation to deplete the most abundant plasma proteins, due to the high dynamic concentration range of proteins. The depletion of high abundant proteins allows to obtain less abundant and, oftentimes, more interesting proteins. However, the removal of the fraction of the high abundant plasma proteins ‐ the depletome ‐ may co‐elute many unintended proteins due to protein‐protein interactions. Little data is available about the depletome and potential protein biomarkers may be lost during this process. To visualize and characterize these proteins, we analyzed the depletome of 20 plasma samples by shotgun mass spectrometry‐based proteomics. Thus, using immunoaffinity depletion followed by 2‐D liquid chromatography coupled to an ion mobility‐enhanced mass spectrometer, our analysis identified that over 100 proteins are co‐eluting with the high abundant fraction. These proteins play roles in several biological processes, such as receptor‐mediated endocytosis, complement activation, and regulation of immune response. This study supports that investigating the depletome is important in the quest for biomarkers.

Quantifying protein aggregation kinetics using electrospray differential mobility analysis

Protein aggregation is a critical concern in bioprocessing, where its presence can result in serious adverse interactions in clinical end-use applications. In this study, an aerosol-based technique, electrospray differential mobility analysis (ES-DMA), was used to quantify thermally-induced protein aggregation kinetics for bovine serum albumin (BSA) and α-chymotrypsinogen A (α-chymo), employing a new methodology to modify the solution for compatibility with the electrospray process. Results are compared orthogonally with asymmetrical-flow field-flow fractionation (AF4), a hydrodynamic separation technique with UV detection. Measurements were conducted over a range of protein concentrations and temperatures. Both techniques successfully resolved the protein monomer and dimer populations, allowing quantification of monomer loss. BSA and α-chymo exhibited second and first order kinetics, respectively, confirming different limiting steps for the two species. The Arrhenius equation yielded activation energies for BSA of (240 ± 20) kJ mol−1 and (190 ± 10) kJ mol−1 by ES-DMA and AF4, respectively. The rates determined by ES-DMA were equal to or slightly faster than those measured by AF4, so instrumental differences were analyzed to identify potential sources of bias. An important factor may be the applicable concentration range for each method; notably, AF4 operates at the mg mL-1 level, while ES-DMA is sensitive at μg mL-1 and therefore requires much smaller samples for analysis (typically several μL are injected). The limitations of each method are detailed in the discussion and demonstrate the importance of orthogonal measurement strategies for the analysis of protein kinetics. ES-DMA provides a potentially useful alternative to size exclusion chromatography to screen the stability of formulation conditions for protein therapeutics; neither ES-DMA nor AF4 rely on column interactions for separation.