Retention Times Of Proteins Research Articles

Advances in Pulmonary Protein Delivery Systems

Protein‐based therapeutics and vaccines play a pivotal role in the realm of biomedical science. Pulmonary administration offers several advantages including rapid adsorption, non‐invasive, increased local drug concentration, and bypassed first‐pass metabolism, thus holding great potential to address multiple unmet medical needs in lung‐related diseases and vaccination. However, the limited success of inhaled proteins in clinical settings highlights the challenges associated with protein stability and the physiological barriers within the respiratory system. To overcome these hurdles, a variety of delivery systems including polymers, liposomes, cell‐derived membranes, and inorganic materials are developed to improve the stability, mucus penetration, retention time, and bioavailability of proteins. With the outbreak of COVID‐19, the pulmonary administration of proteins has drawn great attention. In this review, the design principle, preparation, biomedical application, progress in clinical translation, advantages, and disadvantages of each kind of delivery system are summarized, with an emphasis on carrier materials.

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.

Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level.

PDGF-BB-derived supramolecular hydrogel for promoting skin wound healing

Chronic wounds represent a major challenge to the present healthcare system. In recent decades, many topical therapies have been investigated for the treatment of chronic wounds, including different types of wound dressings, antimicrobial agents, and cell therapy. Platelet-derived growth factor (PDGF) plays an important role in wound healing and has been approved for treatment of wounds related to diabetes mellitus. However, the high cost and short retention time of PDGF protein have limited its wide application. To overcome this challenge, we designed a PDGF-mimicking peptide by connecting PDGF epitope VRKIEIVRKK and self-assembling motif derived from β-amyloid peptide. The resultant peptide can self-assemble into a fibril-rich network and leads to supramolecular hydrogelation with good stability. The hydrophilic epitope can be exposed on the surface of nanofibrils, which might contribute to the binding and activation of PDGF receptors. The forming hydrogel is able to induce the growth and migration of vascular endothelial cells and promote the formation of vascular branches. In the full-thickness skin wounds of healthy mice, after the application of the hydrogel, the density of neovascularization marked by CD31 was greater than that in the control group on Day 3. Larger collagen deposition and a thicker epidermis were observed on Day 12. These results demonstrate that the hydrogel can stimulate collagen deposition and angiogenesis, enhance skin regeneration, and show an excellent therapeutic effect. Taken together, this work not only provides new insight into the design of bioactive peptides but also offers a promising biomaterial for wound healing.

A data-independent acquisition (DIA)-based quantification workflow for proteome analysis of 5000 cells

Data independent acquisition (DIA) has emerged as a powerful proteomic technique with exceptional reproducibility and throughput, and has been widely applied to clinical sample analysis. DIA approaches normally rely on project-specific spectral libraries generated by data dependent acquisition (DDA), requiring extensive off-line fractionation and large amounts of input material. In this study, we aimed to explore the utility of DIA for the analysis of samples with limited quantities. We employed three software tools (DIA-NN, Spectronaut, and EncyclopeDIA) for data analysis and generated three types of libraries, including an experiment-specific library built by DDA analysis of off-line fractions, a FASTA sequence database, and a library generated by gas-phase fractionation (GPF), resulting in eight analysis pipelines. Then we systematically compared the performance of the eight strategies by analyzing the DIA data from HEK293T cell tryptic peptides with sample loads of 500 ng, 100 ng, 20 ng, and 4 ng. The results showed that DIA-NN with GPF-based libraries outperformed the others in protein identification and retention time calibration. Next, we further evaluated the optimized workflow by analyzing the proteome alteration in 5000 peripheral blood mononuclear cells (PBMCs) induced by lipopolysaccharide (LPS) stimulation. As a result, 3179 protein groups were quantified, and functional analysis revealed activation of multiple signaling pathways, e. g., endocytosis, NF-kappa B signaling, and T cell receptor signaling. The results showed the practicability of using DIA for scarce samples, and the established workflow of PBMC analysis could be easily adapted for biomarker discovery, immune status evaluation, and drug response monitoring, especially for diseases involved with dysfunction of the immune system.

Global Identification of Protein Complexes within the Membrane Proteome of Arabidopsis Roots Using a SEC-MS Approach.

Biological processes consist of several consecutive and interacting steps as, for example, in signal transduction cascades or metabolic reaction chains. These processes are regulated by protein-protein interactions and the formation of larger protein complexes, which also occur within biological membranes. To gain a large-scale overview of complex-forming proteins and the composition of such complexes within the cellular membranes of Arabidopsis roots, we use the combination of size-exclusion chromatography and mass spectrometry. First, we identified complex-forming proteins by a retention shift analysis relative to expected retention times of monomeric proteins during size-exclusion chromatography. In a second step we predicted complex composition through pairwise correlation of elution profiles. As result we present an interactome of 963 proteins within cellular membranes of Arabidopsis roots. Identification of complex-forming proteins was highly robust between two independently grown root proteomes. The protein complex composition derived from pairwise correlations of coeluting proteins reproducibly identified stable protein complexes (ribosomes, proteasome, mitochondrial respiratory chain supercomplexes) but showed higher variance between replicates regarding transient interactions (e.g., interactions with kinases) within membrane protein complexes.

Effect of guanidine and arginine on protein-ligand interactions in multimodal cation-exchange chromatography.

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse-grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse-grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435-447, 2017.

Bovine serum albumin adsorbed on eremomycin and grafted on silica as new mixed-binary chiral sorbent for improved enantioseparation of drugs

A new silica-based, mixed-binary chiral sorbent grafted with the macrocyclic antibiotic eremomycin and bovine serum albumin (BSA) was obtained. The sorbent-filled high-performance liquid chromatography column was capable of enantioseparation of racemic drugs, such as profens, in reversed-phase-chromatography mode. The mixed-binary eremomycin-BSA-sorbent showed better capability for profens enantioseparation as compared with a sorbent containing eremomycin only. BSA grafted onto the sorbent surface significantly reduced retention times of other proteins from the analyte solution, and free proteins (including BSA) injected as analytes were not retained on the column, and subsequently eluted with a dead volume. The drastic difference observed in the binding of profens and other proteins using the sorbent was tested for determination and enantioseparation of profens in artificial-urine solutions.

Prediction of protein retention times in hydrophobic interaction chromatography by robust statistical characterization of their atomic-level surface properties.

The correlation between the dimensionless retention times (DRT) of proteins in hydrophobic interaction chromatography (HIC) and their surface properties were investigated. A ternary atomic-level hydrophobicity scale was used to calculate the distribution of local average hydrophobicity across the proteins surfaces. These distributions were characterized by robust descriptive statistics to reduce their sensitivity to small changes in the three-dimensional structure. The applicability of these statistics for the prediction of protein retention behaviour was looked into. A linear combination of robust statistics describing the central tendency, heterogeneity and frequency of highly hydrophobic clusters was found to have a good predictive capability (R2 = 0.78), when combined a factor to account for protein size differences. The achieved error of prediction was 35% lower than for a similar model based on a description of the protein surface on an amino acid level. This indicates that a robust and mathematically simple model based on an atomic description of the protein surface can be used for the prediction of the retention behaviour of conformationally stable globular proteins with a well determined 3D structure in HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:372-381, 2016.

Applicability of the critical chromatography concept to proteomics problems: I. Effect of the stationary phase and the size of the chromatographic column on the dependence of the retention time of peptides and proteins on the amino acid sequence

A theoretical study of the effect of stationary phase parameters on the regularities of the separation of peptides and proteins using a model of critical chromatography BioLCCC and the Theoretical chromatography software created on its basis has been performed. The following problems have been discussed: mechanism of the migration of chromatographic peak along the column and its compression in gradient elution; dependence of the coefficient of protein separation on the column length; inversion of the elution order of peptides at a change of the diameter or length of the column, adsorbent nature, conditions of emergence of inversion; and anomaly of the dependence of the retention time of proteins on the adsorbent activity.

Evaluation of protein drug stability with vitreous humor in a novel ex-vivo intraocular model

The stability of protein therapeutics during the residence time in the vitreous humor (VH) is an important consideration for intra ocular treatment and can possibly impact therapeutic efficacy and/or treatment intervals. Unavailability of the reliable Ex-vivo intravitreal (ExVit) model to estimate protein stability following IVT has driven the research focus to develop such model which can facilitate protein stability estimation before in-vivo experiments. In this manuscript, we have developed and evaluated three ExVit models, namely, ExVit static, semi-dynamic and dynamic. These models were utilized and compared when studying the in-vitro stability of model protein formulations under simulated intraocular conditions using porcine vitreous humor (VH). The ExVit static model exhibited significant precipitation and aggregation of proteins, most likely due to pH change occurred in the VH after isolation. The semi-dynamic model assessed was composed of two compartments i.e., VH- and buffer-compartment which has effectively stabilized the pH of the VH and facilitated the migration of VH degradation products. However, some limitations related to investigation of long-term protein stability were also observed with semi-dynamic model. The dynamic model developed, was comprised of three diffusion controlling barriers (two diffusion controlling membranes and a gel-matrix), which allowed modulation of the diffusion rate of macromolecules. The ability of dynamic model to modulate protein retention time in the VH will overcome the challenges faced by the semi-dynamic model such as long-term stability evaluation.

Behaviour of proteins on reversed-phase supports during high-performance liquid chromatography and prediction of protein retention times on different Zorbax stationary phases

Behaviour of proteins on reversed-phase supports during high-performance liquid chromatography and prediction of protein retention times on different Zorbax stationary phases

The Behavior of Proteins on Reversed-Phase Supports During High-Performance Liquid Chromatography and Prediction of Protein Retention Time on Ymc Columns

Abstract Protein molecules present more complex analytical challenges than conventional low molecular weight pharmaceutical compounds, and thus need powerful analytical approaches for the entire pharmaceutical development and quality control process. We used high-performance liquid chromatography to investigate the reversed-phase chromatographic behavior of eleven proteins (albumin, carbonic anhydrase, cytohrome c, L-glutamic dehydrogenase, enolase, α-lactoglobulin, Lysozyme, myoglobin and ribonuclease. By using a water/organic solvent/trifluoroacetic acid system the influence of experimental parameters was examined, and chromatographic results from two C4- chain-length supports were found to be comparable. The model enables prediction of initial conditions from two experimental data points for different types of reversed-phase columns with water-acetonitrile-TFA, water-methanol-TFA, and water-2-propanol-TFA mobile phases

Dual-mode enhancement of metallothionein protein with cell transduction and retention peptide fusion

Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), have been developed as effective systems for delivering bio-active cargos such as proteins, genes and particles. Further improvements on cell-specific targeting, intracellular organelle targeting and intracellular retention are still necessary to enhance the therapeutic effect of PTD fusion proteins. In order to enhance the cell transduction and retention of anti-oxidative metallothionein protein (MT), MT was recombinantly fused with transcriptional activator (Tat) with or without a short peptide (sMTS) derived from mitochondria malate dehydrogenase (mMDH). Cellular uptake and retention time of fusion protein were significantly increased in the H9c2 cell by sMTS. The Tat–sMTS–MT (TMM) fusion protein protected H9c2 cells more effectively against hypoxia, hyperglycemia and combination compared with Tat–MT (TM) by reducing intracellular ROS level. It maintained the normal blood glucose level over an extended period of time in a streptozotocin-induced diabetic mouse model. PTD–sMTS–MT fusion protein has a potential to be used as a therapeutic protein for the treatment or prevention of diabetes and diabetic complications.

Study on the Adsorption Property of Lysozyme on Weak Cation Exchanger Based on Monodisperse Poly(glycidymethacrylate-co-ethylenedimethacrylate) Beads

A type of weak cation exchanger was prepared based on poly(glycidylmethacrylate-co-ethylenedimethacrylate). The effects of pH and ionic strength on the adsorption behavior were studied, and the results suggested that the adsorption of lysozyme onto a weak cation exchanger is electrostatic interaction, and that the adsorption behavior is in accordance with the Langmuir adsorption model with a correlation coefficient greater than 0.99. It was also found that increasing ionic strength led to a decrease of the adsorption of lysozyme from 49.50 to 28.09 mg/g. Preliminary chromatographic experiments were conducted to test the separation properties of the weak cation exchanger, and the results demonstrated that the retention time of different proteins could be predicted in order of their isoelectric point.

Applicability of the critical-chromatography concept to analysis of proteins: Dependence of retention times on the sequence of amino acid residues in a chain

The BioLCCC model of the chromatographic separation of biomacromolecules, which involves the concepts of the critical chromatography of polymers, is used to describe the experimental data on the separation of proteins on different chromatographic systems. Using phenomenological parameters, i.e., effective adsorption energies of amino acid residues, we predict the effect of the sequence of these residues in the chain on retention times of proteins for reversed phases of different types (C4, C8, C18) in the gradient of a water-acetonitrile binary solvent. It is shown that, in general, the BioLCCC model correctly represents experimental data on the separation of proteins and makes it possible to quantitatively determine the effect of the sequence of amino acid residues on the separation. We show the limits of applicability of the model and explain the universal linear dependence that relates the retention volume and the logarithm of chain length that is observed in the chromatography of peptides and proteins.

Comparison of molecular dynamics simulation methods for amyloid β 1–42 monomers containing d-aspartic acid residues for predicting retention times in chromatography

Molecular dynamics simulations of amyloid β 1–42 containing d-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for d-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the β-structure contents of amyloid β 1–42 peptides obtained by replica exchange molecular dynamics simulations with Onufriev–Bashford–Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid β 1–42 containing d-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form β-sheet structures by themselves, in contrast to the wild-type peptides that form β-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing d-aspartic acid residues.

Surface chemical analysis and chromatographic characterization of polyethylenimine-coated hydroxyapatite with various amount of polyethylenimine

Polyethylenimine (PEI) has been widely used as a coating material to produce stationary phase for ion-exchange chromatography of biomolecules. However, a precise study of the PEI coating fraction has been lacking, despite such quantification being very important for fundamental research as well as identifying further industrial applications.In this study, we produced four types of PEI-coated hydroxyapatite (PEI-HAp) with various fractions of PEI (0.16%, 0.5%, 1.0%, 1.5%) using a spray-drying system to evaluate correlations between coating fractions and the thermochemical or chromatographic behaviors of theses products. The thermal analyses of these matrices showed two exothermic peaks when the PEI coating fraction exceeded 1.0%. The one peak indicated a PEI decomposition peak and the other would indicate bond dissociation of PEI layers formed over the HAp surface as the PEI concentration increased. Furthermore, the chromatographic analysis for the surface chemical characteristics showed the correlation between coating fraction and the retention time of protein or nucleotide. Acidic or phosphorylated proteins were more strongly adsorbed as the PEI coating fraction increased when the initial coating fraction was low, but at fraction exceeding 0.5%, constant retention was observed. The retention time of nucleotides increased in proportion to the fraction of PEI added. The good selectivity of PEI-HAp may be attributable to multifunctional interactions of electrostatic and bare Ca sites on HAp, not just the amino sites of PEI. These precise studies of PEI coating fraction are our original novel contributions, which could be achieved by quantitative consideration using thermal analysis and chromatography.

Monolithic capillary columns synthesized from a single phosphate-containing dimethacrylate monomer for cation-exchange chromatography of peptides and proteins

Monoliths containing phosphoric acid functional groups were synthesized from only one monomer, bis[2-(methacryloyloxy)ethyl] phosphate (BMEP), in 75-μm i.d. UV transparent fused-silica capillaries by photo-initiated polymerization for cation exchange chromatography of peptides and proteins. Various synthetic conditions, including porogen solvents, monomer concentration, and polymerization time, were studied. The hydrophobicities of the resulting monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. These monoliths exhibited low hydrophobicities and relatively low porosities due to their highly cross-linked structures. A dynamic binding capacity (lysozyme) of 73 mg/mL of column volume was measured using the best performing monolith. Synthetic peptides were eluted in approximately 30 min without addition of acetonitrile to the mobile phase, yielding a peak capacity of 28. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. The effects of separation conditions, i.e., mobile phase pH and salt gradient rate, were studied. Good run-to-run reproducibility was achieved with a relative standard deviation (RSD) less than 1.5% for retention times of proteins. The column-to-column retention time reproducibility for peptides was less than 3.5% RSD. A monolithic column was used to follow the deamidation of ribonuclease A. The kinetics of deamidation were founded to be first order with a half life of 195 h. A cytochrome C digest was also separated using a linear gradient of sodium chloride.

Preparation of poly(vinyltetrazole) chain-grafted poly(glycidymethacrylate-co-ethylenedimethacrylate) beads by surface-initiated atom transfer radical polymerization for the use in weak cation exchange and hydrophilic interaction chromatography

A novel stationary phase for weak cation exchange (WCX) and hydrophilic interaction chromatography (HILIC) was prepared with surface-initiated atom transfer radical polymerization (SI-ATRP). Vinyltetrazole was grafted onto the surface of the beads in water medium with the polyglycidylmethacrylate beads (PGMA/EDMA) previously modified with 2-bromoisobutryl bromide as the macromolecule initiators and CuCl as catalyst. The poly(vinyltetrazole)-grafted beads obtained with different atom transfer radical polymerization (ATRP) formulations were tried as chromatographic packings in ion-exchange chromatography. The results showed that the prepared columns could separate the tested proteins with high efficiency and high capacity, and the retention time of protein had a positive relationship with increasing the chain lengths of the grafted poly(vinyltetrazole) (PVT). The prepared column was also found to be able to separate nucleosides by hydrophilic interaction chromatographic mode.

Ion Exchange Chromatographic Behavior of a Homologous Cytochrome C Variant Library Obtained by Controlled Succinylation

A cytochrome C homologous library with varying charge distribution was employed to study the effects of protein surface modification on protein retention in cation exchange chromatography. Varying quantities of succinic anhydride were added to horse cytochrome c to control the degree of succinylation of protein surface lysine residues. Cation exchange chromatography was then carried out using a succinylated protein mixture containing primarily single lysine modifications and the collected fractions were evaluated using direct infusion and tryptic digest–mass spectrometry to determine the degree of succinylation and the sites of modification on the protein surface. Electrostatic potential (EP) maps of the native protein and the variants were generated to provide insight into the elution order of the variants and the results indicated that there were two major binding regions on the protein surface. Variants showing the largest change in retention time as compared to the native protein had modified residues within the binding regions. On the other hand, variants which showed a smaller change in protein retention time had succinylated residues at locations which were not part of the two distinct binding regions. This study demonstrates that the use of controlled protein surface modification offers a convenient means of studying the relationship between protein surface properties and chromatographic binding affinity.