Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap.

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and ß-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Production of highly purified fractions of α-lactalbumin and ß-lactoglobulin from cheese whey using high hydrostatic pressure.

Despite extensive research on the topic, valorization of dairy by-products remains challenging. Cheese whey is of particular interest because it contains valuable proteins such as α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). However, selective fractionation of these 2 proteins into pure fractions is complex because of their similar molecular weights. In this study, we proposed an innovative protein separation strategy based on coupling high hydrostatic pressure (HHP) with acidification of whey at pH 4.6. We investigated the effect of single-cycle HHP (600 MPa) for 5, 10, and 15 min and multiple-cycle HHP (1-3 cycles of 5 min at 600 MPa) on α-LA and ß-LG fractionation from cheese whey at initial pH (control, pH 6.66) and acidified to pH 4.6. All pressurization conditions with acidified whey induced a drastic aggregation of ß-LG compared with control whey. The highest degrees of purification (75 and 98%, respectively) and yields (95 and 88%, respectively) of α-LA and ß-LG were obtained with the application of single-cycle HHP treatment of acidified whey at pH 4.6 at 600 MPa for 5 min. Our results showed the strong potential of using HHP as an innovative tool for the fractionation of valuable proteins such as α-LA from cheese whey.

Impact of Preheating Temperature on the Separation of Whey Proteins When Combined with Chemical or Bipolar Membrane Electrochemical Acidification.

Separation of α-lactalbumin and ß-lactoglobulin improves their respective nutritional and functional properties. One strategy to improve their fractionation is to modify their pH and ionic strength to induce the selective aggregation and precipitation of one of the proteins of interest. Electrodialysis with bipolar membrane (EDBM) is a green process that simultaneously provides acidification and demineralization of a solution without adding any chemical compounds. This research presents the impact on whey proteins separation of different preheating temperatures (20, 50, 55 and 60 °C) combined with EDBM or chemical acidification of 10% whey protein isolate solutions. A ß-lactoglobulin fraction at 81.8% purity was obtained in the precipitate after EDBM acidification and preheated at 60 °C, representing a recovery yield of 35.8%. In comparison, chemical acidification combined with a 60 °C preheating treatment provides a ß-lactoglobulin fraction at 70.9% purity with a 11.6% recovery yield. The combination of EDBM acidification with a preheating treatment at 60 °C led to a better separation of the main whey proteins than chemical acidification.

Isolation and Self-Association Studies of Beta-Lactoglobulin.

The aim of this study was to investigate isolated ß-lactoglobulin (ß-LG) from the whey protein isolate (WPI) solution using the column chromatography with SP Sephadex. The physicochemical characterization (self-association, the pH stability in various salt solutions, the identification of oligomeric forms) of the protein obtained have been carried out. The electrophoretically pure ß-LG fraction was obtained at pH 4.8. The fraction was characterized by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) technique. The use of the HCCA matrix indicated the presence of oligomeric ß-LG forms, while the SA and DHB matrices enabled the differentiation of A and B isoforms in the sample. The impact of sodium chloride, potassium chloride, ammonium sulfate, and sodium citrate in dispersion medium on ß-LG electrophoretic stability in solution was also studied. Type of the dispersion medium led to the changes in the isoelectric point of protein. Sodium citrate stabilizes protein in comparison to ammonium sulfate. Additionally, the potential of capillary electrophoresis (CE) with UV detection using bare fused capillary to monitor ß-LG oligomerization was discussed. Obtained CE data were further compared by the asymmetric flow field flow fractionation coupled with the multi-angle light scattering detector (AF4-MALS). It was shown that the ß-LG is a monomer at pH 3.0, dimer at pH 7.0. At pH 5.0 (near the isoelectric point), oligomers with structures from dimeric to octameric are formed. However, the appearance of the oligomers equilibrium is dependent on the concentration of protein. The higher quantity of protein leads to the formation of the octamer. The far UV circular dichroism (CD) spectra carried out at pH 3.0, 5.0, and 7.0 confirmed that ß-sheet conformation is dominant at pH 3.0, 5.0, while at pH 7.0, this conformation is approximately in the same quantity as α-helix and random structures.

Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics.

A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (1D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (2D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on-off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

An automated food protein isolation approach on preparative scale by two-dimensional liquid chromatography with active modulation interface.

In this work, an automated 2D-LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size-exclusion column, packed with 3 µm ultrapure silica particles was used. An RP column based on core-shell technology was used for the second dimension separation. A standard mixture of BSA, ß-lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single-chromatographic analysis, a protein amount of 50 µg for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large-scale investigations in biological systems.

Fast reversed-phase liquid chromatographic separation of proteins by flow-through poly(styrene-co-divinylbenzene) microspheres.

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High-performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow-through poly(styrene-co-divinylbenzene) microspheres characteristic of the binary pores, i.e. flow-through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene-co-divinylbenzene) microspheres were suitable as the reversed-phase stationary phase for separation of proteins. For the high permeability of the poly(styrene-co-divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ∼2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0-99.4%. The proposed column had good pH stability of 1-13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio-sample separation. The flow-through poly(styrene-co-divinylbenzene) microspheres were promising for fast separation of large molecules.

Comparison of selective hydrolysis of α-lactalbumin by acid Protease A and Protease M as alternative to pepsin: potential for ß-lactoglobulin purification in whey proteins.

The experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving ß-lactoglobulin (ß-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for ß-Lg isolation.

Poly(norepinephrine)-coated open tubular column for the separation of proteins and recombination human erythropoietin by capillary electrochromatography.

Recombinant human erythropoietin is an important therapeutic protein with high economic interest due to the benefits provided by its clinical use for the treatment of anemias associated with chronic renal failure and chemotherapy. In this work, a poly(norepinephrine)-coated open tubular column was successfully prepared based on the self-polymerization of norepinephrine under mild alkaline condition, the favorable film forming and easy adhesive properties of poly(norepinephrine). The poly(norepinephrine) coating was characterized by scanning electron microscopy and measurement of the electro-osmotic flow. The thickness of the coating was about 431 nm. The electrochromatographic performance of the poly(norepinephrine)-coated open tubular column was evaluated by separation of proteins. Some basic and acidic proteins including two variants of bovine serum albumin and two variants of ß-lactoglobulin achieved separation in the poly(norepinephrine)-coated open tubular column. More importantly, the column demonstrated separation ability for the glycoforms of recombinant human erythropoietin. In addition, the column demonstrated good repeatability with the run-to-run, day-to-day, and column-to-column relative standard deviations of migration times of proteins less than 3.40%.

[Optimizing the detection of bovine milk ß-Lactoglobulin with protein chip].

OBJECTIVE: To optimize the conditions of protein chip assay for bovine milk ß-Lactoglobulin( ß-Lg). METHODS: A microarrayer was used for printing anti-ß-Lg as antibody I on each 3-dimensional-slide, another antis ß-Lg antibody was used as detection antibody II and goat antibody coupled to Cy3 was used as antibody III. The standard ß-Lg was detected by double antibody sandwich technique. RESULTS: Mouse monoclonal ß-Lg antibody66# was chosen as the probe and contact printing as the printing method. The range between 42 and 92 spots was chosen as the basic printing condition. The concentration of ß-Lg probes was 0. 5 mg / mL. The ß-Lg detection antibody titre was 1∶2000. One percent no protein blocking solution was choosen as the blocking buffer. The lower detection limit and the biological detection limit of ß-Lg were 17. 54 ng / m L and 55. 31 ng / m L respectively. The linear range was determined according to the S type curve of ß-Lg and the best fitting models and standard curve were established for ß-Lg( R~2=0. 9993). CONCLUSION: The study optimizes conditions of a quantitative analysis system for measurement of ß-Lg with protein chip, thus establishing the protein chip platform for quantitative detection of bovine milk ß-Lactoglobulin.

Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography.

Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO2) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO2 coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti(4+) to boost the nucleation and growth of TiO2 film. The as-prepared TiO2 coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO2 coating was investigated. The TiO2 coated OT column was used for successful separation of two variants of ß-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white.

On-chip intermediate LED-IF-based detection for the control of electromigration in multichannel networks.

Monitoring analytes during the transfer step from the first to the second dimension in multidimensional electrophoretic separations is crucial to determine and control the optimal time point for sample transfer and thus to avoid band broadening or unwanted splitting of the sample band with consequent sample loss. A spatially resolved intermediate on-chip LED-induced fluorescence detection system was successfully implemented for a hybrid capillary-chip glass interface. The setup includes a high-power 455-nm LED prototype as an excitation light source and a linear light fiber array consisting of 23 light fibers with a diameter of 100 µm for spatially resolved fluorescence detection in combination with a push-broom imager for hyperspectral detection. Using a basic FITC solution, the linear working range was determined to be 0.125 to 25 µg/ml for a single light guide and the absolute detection limit was 0.04 fmol at a signal-to-noise ratio of 4. With the setup presented here, labeled ß-lactoglobulin focused via capillary isoelectric focusing was detectable on-chip with a sufficient intensity to monitor the analyte band transfer in the glass-chip interface demonstrating its applicability for full or intermediate on-chip detection.

Detecting ß-Casein Variation in Bovine Milk.

In bovine species, ß-casein (ß-CN) is characterized by genetic polymorphism. The two most common protein variants are ß-CN A² (the original one) and A¹, differing from A² for one amino acid substitution (Pro67 to His67). Several bioactive peptides affecting milk nutritional properties can originate from ß-CN. Among them, ß-casomorphin-7 (BCM7) ranging from amino acid 60 to 66 can be released more easily from ß-CN variants carrying His67 (A¹ type) instead of Pro67 (A² type). Nowadays, "A2 milk" is produced in different countries claiming its potential benefits in human health. The aim of this study was to further develop and apply an isoelectric focusing electrophoresis (IEF) method to bulk and individual milk samples in order to improve its use for ß-CN studies. We succeeded in identifying A2 milk samples correctly and quantifying the percentage of A², A¹, and B variants in bulk samples not derived from A2 milk as well as in individual milk samples. The method allows us to quantify the relative proportion of ß-CN variants in whole milk without eliminating whey protein by acid or enzymatic precipitation of caseins. The aim of this study was also to study the different behavior of ß-CN and ß-lactoglobulin (ß-LG) in the presence of trichloroacetic acid (TCA). The higher sensitivity of ß-CN to TCA allows quantifying ß-CN variants after TCA fixation because ß-LG is not visible. Monitoring ß-CN variation in cattle breeds is important in order to maintain a certain balance between Pro67 and His67 in dairy products. Overall, the debate between A1 and A2 milk needs further investigation.

Complex equilibria, speciation, and heteroprotein coacervation of lactoferrin and ß-lactoglobulin.

There has been a resurgence of interest in complex coacervation, a form of liquid-liquid phase separation (LLPS) in systems of oppositely charged macroions, but very few reports describe the somewhat anomalous coacervation between acidic and basic proteins, which occurs under very narrow ranges of conditions. We sought to identify the roles of equilibrium interprotein complexes during the coacervation of ß-lactoglobulin dimer (BLG2) with lactoferrin (LF) and found that this LLPS arises specifically from LF(BLG2)2. We followed the progress of complexation and coacervation as a function of r, the LF/BLG molar ratio, using turbidity to monitor the degree of coacervation and proton release and dynamic light scattering (DLS) to assess the stoichiometry and abundance of complexes. Isothermal titration calorimetry (ITC) showed that initial complex formation is endothermic, but a large exotherm related to coacervate formation obscured other regions. On the basis of turbidimetry, proton release, and DLS, we propose a speciation diagram that presents the abundance of various complexes as a function of r. Although multiple species could be simultaneously present, distinct regions could be identified corresponding to equilibria among particular protein pairs.

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and ß-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin.

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for ß-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.

Protein-selective coacervation with hyaluronic acid.

Selective coacervation with hyaluronic acid (HA), a biocompatible and injectable anionic polysaccharide, was used to isolate a target protein, bovine serum albumin (BSA), with 90% purity from a 1:1 mixture with a second protein of similar pI, ß-lactoglobulin (BLG). This separation was attributed to the higher HA-affinity of BSA, arising from its more concentrated positive domain. The values of pH corresponding respectively to the onset of complex formation, coacervation, precipitation, and redissolution (pH(c), pHϕ, pH(p), and pH(d)) were determined as a function of ionic strength I. These pH values were related to critical values of protein charge, Z, and their dependence on I provided some insights into the mechanisms of these transitions. The higher polyanion binding affinity of BSA, deduced from its higher values of pH(c), was confirmed by isothermal titration calorimetry (ITC). Confocal laser microscopy clearly showed time-dependent coalescence of vesicular droplets into a continuous film. Comparisons with prior results for the polycation poly(diallyldimethylammonium chloride) (PDADMAC) show reversal of protein selectivity due to reversal of the polyelectrolyte charge. Stronger binding of both proteins to PDADMAC established by ITC may be related to the higher chain flexibility and effective linear charge density of this polycation.

ß-Lactoglobulin-based aerogels: Facile preparation and sustainable removal of organic contaminants from water.

Economically and efficiently removing organic pollutants from water is still a challenge in wastewater treatment. Utilizing environmentally friendly and readily available protein-based natural polymers to develop aerogels with effective removal performance and sustainable regeneration capability is a promising strategy for adsorbent design. Here, a robust and cost-effective method using inexpensive ß-lactoglobulin (BLG) as raw material was proposed to fabricate BLG-based aerogels. Firstly, photocurable BLG-based polymers were synthesized by grafting glycidyl methacrylate. Then, a cross-linking reaction, including photo-crosslinking and salting-out treatment, was applied to prepared BLG-based hydrogels. Finally, the BLG-based aerogels with high porosity and ultralight weight were obtained after freeze-drying. The outcomes revealed that the biocompatible BLG-based aerogels exhibited effective removal performance for a variety of organic pollutants under perfectly quiescent conditions, and could be regenerated and reused many times via a simple and rapid process of acid washing and centrifugation. Overall, this work not only demonstrates that BLG-based aerogels are promising adsorbents for water purification but also provides a potential way for the sustainable utilization of BLG.

Identification and characterization of cow's milk proteins from the rat intestinal lymph using a proteomic strategy.

Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides, and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow's milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by 1D-SDS-PAGE, digested in-gel based on molecular weight, and identified using nano-LC-MS/MS. In the postprandial rat intestinal lymph, nine bovine-specific proteins (false discovery rate ≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were highly abundant proteins in the milk, such as ß-lactoglobulin and caseins. Seven of the nine identified bovine-specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross-barrier transport or translocation.

Direct AFM force mapping of surface nanoscale organization and protein adsorption on an aluminum substrate.

We investigate the nanoscale organization of a superficially hydroxylated Al substrate and its effect on subsequent protein adsorption using atomic force microscopy (AFM). For this purpose we used a mode which allows a direct mapping of a variety of surface properties (adhesion, elasticity, dissipation, etc.) to be probed simultaneously with topographical images. The hydroxylation treatment leads to a drastic modification of the surface morphology, owing to the formation of AlOOH compounds. In air, AFM images revealed the formation of regular nanorod-like structures randomly distributed, inducing the appearance of nanoporous domains on the surface. In buffer solution, prior to the adsorption of proteins, the surface nanoscale organization is preserved, mainly due to the chemical stability of AlOOH compounds under these conditions. The adsorption of proteins on the obtained nanostructured surface was performed using either a globular (ß-lactoglobulin) or a fibrillar (collagen) protein and by modulating the adsorbed amount through the incubation time or the concentration of proteins in solution. At low amounts, collagen adsorbs on the whole surface without preferential localization. The surface topography remains similar to the bare surface, while significant changes were evidenced on adhesion and elasticity maps. This is due to the fact that the surface became adhesive and less stiff, owing to the presence of a soft and hydrated protein layer. By contrast, ß-lactoglobulin tends to diffuse into the nanoporous domains, leading to their filling up, and the surface is blurred with a thick and dense protein layer upon increasing the amount of adsorbed molecules. Our findings demonstrate the interest in using AFM for surface mapping to investigate the mechanism of protein adsorption at the nanoscale on materials with high surface roughness.