Automation and low-cost proteomics for characterization of the protein corona: experimental methods for big data.

Nanoparticles used in biological settings are exposed to proteins that adsorb on the surface forming a protein corona. These adsorbed proteins dictate the subsequent cellular response. A major challenge has been predicting what proteins will adsorb on a given nanoparticle surface. Instead, each new nanoparticle and nanoparticle modification must be tested experimentally to determine what proteins adsorb on the surface. We propose that any future predictive ability will depend on large datasets of protein-nanoparticle interactions. As a first step towards this goal, we have developed an automated workflow using a liquid handling robot to form and isolate protein coronas. As this workflow depends on magnetic separation steps, we test the ability to embed magnetic nanoparticles within a protein nanoparticle. These experiments demonstrate that magnetic separation could be used for any type of nanoparticle in which a magnetic core can be embedded. Higher-throughput corona characterization will also require lower-cost approaches to proteomics. We report a comparison of fast, low-cost, and standard, slower, higher-cost liquid chromatography coupled with mass spectrometry to identify the protein corona. These methods will provide a step forward in the acquisition of the large datasets necessary to predict nanoparticle-protein interactions.

Cracking Proteoform Complexity of Ovalbumin with Anion-Exchange Chromatography-High-Resolution Mass Spectrometry under Native Conditions.

Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 600 different types of posttranslational modifications are known, many of them being of extremely low abundance, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterization. Here, we report the development of a novel pH gradient-based anion-exchange chromatography method, which can be directly interfaced to Orbitrap-based mass spectrometry for the comprehensive characterization of proteoforms at the intact protein level under native conditions. The analysis of four different proteins demonstrates outstanding chromatographic selectivity, while the mass spectra obtained are of excellent quality enabling the identification of proteoforms, including near isobaric variants, spanning 4 orders of magnitude in abundance. An in-depth analysis of ovalbumin from chicken egg white yields the identification and relative quantification of more than 150 different proteoforms, including fragmented and dimeric forms. More than 20 different ovalbumin charge variants together with their glycoform distributions are identified and quantified, many of which have not been reported previously.

Increasing the amount of phosphoric acid enhances the suitability of Bradford assay for proteomic research.

The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced.

Orientationally Fabricated Zwitterionic Molecularly Imprinted Nanocavities for Highly Sensitive Glycoprotein Recognition.

Glycoprotein recognition has recently gained a lot of attention, since glycoproteins play important roles in a diverse range of biological processes. Robustly synthesized glycoprotein receptors, such as molecularly imprinted polymers (MIPs), which can be easily and sustainably handled, are highly attractive as antibody substitutes because of the difficulty in obtaining high-affinity antibodies specific for carbohydrate-containing antigens. Herein, molecularly imprinted nanocavities for glycoproteins have been fabricated via a bottom-up molecular imprinting approach using surface-initiated atom transfer radical polymerization (SI-ATRP). As a model glycoprotein, ovalbumin was immobilized in a specific orientation onto a surface plasmon resonance sensor chip by forming a conventional cyclic diester between boronic acid and cis-diol. Biocompatible polymer matrices were formed around the template molecule, ovalbumin, using SI-ATRP via a hydrophilic comonomer, 2-methacryloyloxyethyl phosphorylcholine, in the presence of pyrrolidyl acrylate (PyA), a functional monomer capable of electrostatically interacting with ovalbumin. The removal of ovalbumin left MIPs with binding cavities containing boronic acid and PyA residues located at suitable positions for specifically binding ovalbumin. Careful analysis revealed that strict control over the polymer significantly improved sensitivity and selectivity for ovalbumin recognition, with a limit of detection of 6.41 ng/mL. Successful detection of ovalbumin in an egg white matrix was demonstrated to confirm the practical utility of this approach. Thus, this strategy of using a polymer-based recognition of a glycoprotein through molecularly imprinted nanocavities precisely prepared using a bottom-up approach provides a potentially powerful approach for detection of other glycoproteins.

Simultaneous determination of proteins in microstructured optical fibers supported by chemometric tools.

A new perspective on the relevant problem-creating simple, rapid, and efficient protein sensors based on microstructured optical fibers using a simple homogeneous analysis format-was proposed. Commercially available long-period grating hollow core microstructured optical fibers (LPG HCMOF) were used to determine bovine serum albumin (BSA) and albumin from chicken eggs (OVA) in binary mixtures as well as immunoglobulin G (IgG) in the presence of BSA and OVA. LPG HCMOF transmission spectra allowed the detection of both BSA and OVA up to 10 mg/mL with LOD as low as 0.1 and 0.8 µg/mL, respectively. Partial least squares regression (PLS) was utilized for modeling of LPG HCMOF spectral data and quantitative analysis of BSA, OVA, total protein, and IgG in binary and ternary mixtures. Rather high coefficients of determination (R2) and low root mean square error for the calibration (RMSEC) (15%) and prediction (RMSEP) (20%) were obtained for all PLS models. The proposed approach was tested in the analysis of BSA in spiked horse blood hemolyzed (HBH). The results demonstrated the functionality of the proposed approach and offered the opportunity for the creation of a wide range of sensors for protein determination in complex mixtures. Graphical abstract.

Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Effects of dietary methionine on productivity, reproductive performance, antioxidant capacity, ovalbumin and antioxidant-related gene expression in laying duck breeders.

The study investigated whether dietary methionine (Met) affects egg weight and antioxidant status through regulating gene expression of ovalbumin (OVAL), nuclear factor erythroid 2 like 2 (Nrf2) and haem oxygenase 1 (HO-1) in laying duck breeders. Longyan duck breeders (n 540, 19 weeks) were randomly assigned to six treatments with six replicates of fifteen birds each. Breeders were fed diets with six Met levels (2·00, 2·75, 3·50, 4·25, 5·00 and 5·75 g/kg) for 24 weeks. The egg weight (g), egg mass (g/d), feed conversion ratio, hatchability, 1-d duckling weight, albumen weight, albumen proportion and OVAL mRNA level improved with dietary Met levels, whereas yolk proportion decreased (P<0·05). The weight of total large yellow follicles increased linearly (P<0·001) and quadratically (P<0·05) with dietary Met concentration, and their weight relative to ovarian weight showed a linear (P<0·05) effect. Dietary Met level had a linear (P<0·05) and quadratic (P<0·001) effect on the gene expression of glutathione peroxidase (GPX1), HO-1 and Nrf2, and quadratically (P<0·05) increased contents of GPX and total antioxidant capacity (T-AOC) in liver of duck breeders. In addition, maternal dietary Met enhanced gene expression of GPX1, HO-1 and Nrf2, increased contents of GPX and T-AOC and reduced carbonylated protein in the brains of hatchlings. Overall, dietary Met concentration affected egg weight and albumen weight in laying duck breeders, which was partly due to gene expression of OVAL in oviduct magnum. A diet containing 4·0 g Met/kg would achieve optimal hepatic GPX1 and Nrf2 expression, maximise the activity of GPX and minimise lipid peroxidation.

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

[Development of a Method for Simultaneous Analysis of Egg and Milk Major Allergens in Beverage Products Using High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry].

A method for simultaneous analysis of egg and milk allergens using LC-QTOF-MS was developed. The proteins measured were α-casein, ß-lactoglobulin, and ovalbumin, which are the main protein allergens in milk and eggs. The proteins were digested using trypsin, and the digests were analyzed by LC-QTOF-MS. Sixteen peaks were detected that confirmed the amino acid sequences of the digests, and a MRM method with high resolution (MRM-HR) using product ions of these peaks was applied for quantification. Next, validation studies were performed using beverage products to which milk and egg standard protein solutions had been added. Good linearity was achieved over the concentration range of 1.25 to 20 µg/g of milk and egg protein, and acceptable reproducibility and accuracy were obtained at 10 µg/g. Moreover, good agreement was also observed between LC-QTOF-MS and ELISA. These findings suggest that this LC-QTOF-MS method may be useful for determining the milk and egg protein contents of beverages.

Development of a mass spectrometry immunoassay for unambiguous detection of egg allergen traces in wines.

A mass spectrometry immunoassay (MSIA) specifically designed for the detection of egg allergens in wines is described. MSIA is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentrations in the low microgram-per-milliliter range. A simple protocol was devised consisting of a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on MSIA customized disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit, LOD and LOQ values as low as 0.01 and 0.03 µg/mL, respectively, and within-day precision of 18% should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition, compared to other immunoassays, the present approach boasts the unquestionable advantage of providing an unambiguous identification of the target protein by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.

Development of C3-Symmetric Tris-Urea Low-Molecular-Weight Gelators.

This article describes recent developments in C3 -symmetric tris-urea low-molecular-weight gelators and their applications. The C3 -symmetric tris-ureas are excellent frameworks to form supramolecular polymers through noncovalent interactions. In organic solvents, hydrophobic tris-ureas form supramolecular gels. Amphiphilic tris-ureas form supramolecular gels in aqueous media. Functional supramolecular gels were prepared by introducing appropriate functional groups into the outer sphere of tris-ureas. Supramolecular hydrogels obtained from amphiphilic tris-ureas were used in the electrophoresis of proteins. These electrophoreses results showed several unique characteristics compared to typical electrophoreses results obtained using polyacrylamide matrices.

Preparation of magnetic glycoprotein-imprinted nanoparticles with dendritic polyethyleneimine as a monomer for the specific recognition of ovalbumin from egg white.

Glycoproteins are crucial in massive physiological events and clinical application. It is necessary to prepare new materials to isolate the specific glycoprotein. New and simple core-shell molecularly imprinted polymers were prepared by surface imprinting. The polymers are synthesized with magnetic nanoparticles as the core, water-soluble dendritic polyethyleneimine as the monomer and the ovalbumin as the template. The prepared imprinted polymers showed thin imprinted shell, biocompatibility and superparamagnetic properties. The resultant materials exhibited fast kinetics, high adsorption capacity, perfect selectivity and reusability. More important, they can absorb the template glycoprotein from the neutral solution and successfully be applied to recognize the ovalbumin from egg white, which means that they can provide an alternate method to isolate glycoprotein from bodily fluids.

Aggregation of egg white proteins with pulsed electric fields and thermal processes.

BACKGROUND: Pulsed electric field (PEF) processing is progressing towards application for liquid egg to ensure microbial safety. However, it usually causes protein aggregation, and the mechanism is still unclear. In this study, egg white protein was applied to investigate the changes in protein structure and mechanism of aggregates formation and a comparison was made with thermal treatment. RESULTS: Soluble protein content decreased with the increase of turbidity after both treatments. Fluorescence intensity and free sulfhydryl content were increased after being treated at 70 °C for 4 min. Less-remarkable changes of hydrophobicity were observed after PEF treatments (30 kV cm(-1) , 800 µs). Soluble and insoluble aggregates were observed by thermal treatment, and disulfide bonds were the main binding forces. The main components of insoluble aggregates formed by thermal treatment were ovotransferrin (30.58%), lysozyme (18.47%) and ovalbumin (14.20%). While only insoluble aggregates were detected during PEF processes, which consists of ovotransferrin (11.86%), lysozyme (21.11%) and ovalbumin (31.07%). Electrostatic interaction played a very important role in the aggregates formation. CONCLUSION: PEF had a minor impact on the structure of egg white protein. PEF had insignificant influence on heat-sensitive protein, indicating that PEF has potential in processing food with high biological activity and heat sensitive properties. © 2015 Society of Chemical Industry.

Magnetic beads-based electrochemical immunosensor for monitoring allergenic food proteins.

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222nM and a detection limit of 5nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.

Distyrylbenzene-aldehydes: identification of proteins in water.

Three different, water soluble, aldehyde-appended distyrylbenzene (DSB) derivatives were prepared. Their interaction with different albumin variants (human, porcine, bovine, lactalbumin, ovalbumin) was investigated (pH 11). All three fluorophores exhibit graded, protein-dependent fluorescence turn-on at slightly differing wavelengths. Linear discriminant analysis (LDA) differentiated all of the investigated albumins and was used to discern commercially available protein shakes. The three DSB derivatives barely react with the constituting amino acids but cysteine. In the proteins significant fluorescence signals are generated, probably due to a combination of imine/N,S-aminal formation and hydrophobic interactions between the DSBs and the proteins.

Quantification of proteins using (13)C7-labeled and unlabeled iodoacetanilide by nano liquid chromatography/nanoelectrospray ionization and by selected reaction monitoring mass spectrometry.

The combination of cysteine-specific modifiers, iodoacetanilide (IAA) and (13)C7-labeled iodoacetanilide ((13)C7-IAA), has been applied to absolute quantification of proteins. The selected reaction monitoring (SRM) with the use of nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS) analysis was applied to precise quantification of three commercial proteins. Good correlation was observed between the theoretical ratios and observed ratios for all these proteins both in a simple buffer solution and in a complex protein environment. Due to efficient tagging, this method does not require separate synthesis of isotope-labeled peptides for the SRM studies. Therefore, this method is expected to be a useful tool for proteomics research.

Rapid and label-free detection of egg allergen traces in wines by surface plasmon resonance biosensor.

The development of a surface plasmon resonance (SPR)-based biosensor tailored to the fast detection of egg-related fining allergens in wines is herein described. Ovalbumin (OVA) was chosen as the target protein to be monitored due to its highest abundance in the egg white powder, a typical fining agent used by the winery industry to promote wine clarification. A direct assay was designed, basing on the use of polyclonal anti-OVA antibody as bio-specific receptor. With the aim of optimizing the assay conditions, different parameters able to influence the final biosensor response were carefully investigated (i.e., pH, ionic strength, and additional surfactant concentration). After the fine tuning of these parameters, the assay was tested in the direct analysis of OVA in commercial wines artificially contaminated with egg white powder at different concentration levels in order to assess the reliability of the biosensor in detecting traces of OVA in complex matrices. The devised assay allowed to trace, in a short analysis time and with a minimal sample pre-treatment required, the presence of egg allergens at the lowest concentration comprised between 0.03 and 0.2 µg/mL. Finally, the response provided by the developed biosensor was correlated with an established liquid chromatography mass spectrometry (LC-MS) method developed in our laboratories, and performances of both approaches were assessed for the fast monitoring of egg allergen contamination in fined wines.

Interactive effects of vitamin D3 and strontium on performance, nutrient retention and bone mineral composition in laying hens.

BACKGROUND: Strontium is currently prescribed for patients with osteoporosis to increase bone density and reduce bone fractures but its relevance in animal nutrition is obscure. In order to investigate the effect of supplemental strontium and vitamin D3 on performance, egg quality and skeletal integrity in poultry a total of 108 laying hens, 99 weeks of age, were fed three levels of strontium (0, 500, 1000 mg kg(-1) ) and two levels of vitamin D3 (2500, 5,000 iu kg(-1)) over a 12-week period. RESULTS: There was an improvement (P < 0.05) in egg production and feed conversion efficiency with strontium at 500 mg kg(-1) and a significant increase in egg weight in those hens fed additional vitamin D3 . Supplemental strontium increased phosphorus, sodium and strontium retention in birds fed 2500 iu D3 kg(-1) but reduced phosphorus, sodium and strontium retention in birds fed 5000 iu D3 kg(-1), resulting in an interaction (P < 0.01) between strontium and vitamin D3 . Addition of 5000 iu D3 kg(-1) increased egg weight (P < 0.05); predominantly by increased albumen content (P < 0.05), whereas strontium supplementation reduced egg weight (P < 0.001). Similarly, 5000 iu kg(-1) D3 increased apparent metabolizable energy (P < 0.05); in contrast, strontium supplementation reduced (P < 0.05) apparent metabolizable energy. CONCLUSION: The addition of 500 mg kg(-1) strontium significantly improved egg production and feed efficiency; however, further investigation needs to be undertaken to refine the optimum level of strontium required to maximize hen performance. The interrelationship between strontium and vitamin D3 requires further exploratory study.

Impact of protein concentration on the determination of thiolic groups of ovalbumin: a size exclusion chromatography-chemical vapor generation-atomic fluorescence spectrometry study via mercury labeling.

We optimized a hyphenated system based on size exclusion chromatography coupled to a microwave/UV mercury oxidation system and an atomic fluorescence detector (SEC-CVG-AFS) for the online oxidation of free and protein-complexed p-hydroxymercuribenzoic acid (pHMB) without the employment of chemical oxidizing agents. This system has been applied to the study of labeling of thiolic groups of native ovalbumin (OVA) as a function of protein concentration. We found that the protein concentration strongly affects the species distribution of OVA, the number of thiolic groups titrated in each species, and thus, the accuracy in the determination of the total number of thiolic groups. The amount of titrated sulfhydryl groups in the protein concentration range investigated (5-100 µmol L(-1)) varied from 2.40 ± 0.01 to 1.85 ± 0.05 for the monomeric form of OVA and from 4.63 ± 0.01 to 5.63 ± 0.05 for the total OVA, which represents more than four theoretical number of reduced Cys. This information is important from the analytical point of view because it suggests that, unless to operate with diluted concentration of protein, the number of titrated thiolic groups results from both the aspecific interaction of the probe with aggregates species and to the specific bond of the probe with the accessible -SH groups.

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS-PAGE.

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and ß-casein, 62 ng of ovalbumin, phosvitin, and κ-casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.