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.

Direct separation and purification of α-lactalbumin from cow milk whey by aqueous two-phase flotation of thermo-sensitive polymer/phosphate.

BACKGROUND: α-lactalbumin (α-La) is of great interest to the industry as a result of its excellent functional properties and nutritional value. Aqueous two-phase flotation (ATPF) of thermo-sensitive polymer poly (ethylene glycol-ran-propylene glycol) monobutyl ether (UCON) and KH2 PO4 was applied to directly separate and purify α-La from milk whey, which was purposed to simplify the production process and reduced cost of production. RESULTS: The effect of ATPF composition and operating parameters on the flotation efficiency (E) and purity of α-La were investigated. The optimal conditions included 2 min of premixing time, 30 mL min-1 flow velocity and 20 min of flotation time, whereas the composition conditions comprised 35.0 mL 0.18 g mL-1 phosphate solution (containing 10% (cow milk whey/salt solution, v/v) cow milk whey, 50 ppm defoamer and 2 g NaCl) and 5.0 mL of 40% (w/w) UCON solution. Under the optimal conditions, E of α-La was 95.67 ± 1.04% and purity of α-La was 98.78 ± 1.19%. UCON was recovered by a thermally-induced phase separation and reused in next ATPF process without reducing E of α-La. Purified α-La was characterized by several key technologies. The results indicated that α-La in cow milk whey could be directly separated and purified by the ATPF and the purity was satisfactory. Moreover, it was suggested there was no obvious structure difference between the α-La separated by ATPF and the α-La standard. CONCLUSION: The present study enabled the recycling of UCON, providing an effective, economically viable and environmentally friendly approach for the separation and purification of protein. © 2021 Society of Chemical Industry.

Antioxidant and antibacterial activities, interfacial and emulsifying properties of the apo and holo forms of purified camel and bovine α-lactalbumin.

The antioxidant and antibacterial activities of camel and bovine α-lactalbumin (α-La) in both calcium-loaded (holo) and calcium-depleted (apo) forms were investigated and compared. Antioxidant assay showed that camel and bovine α-La exhibited significant Ferric-reducing antioxidant power (FRAP), ferrous iron-chelating activity (FCA) and antiradical activities especially in their apo form. Camel apo α-La also exhibited attractive antibacterial activities against Gram-negative bacteria (Pseudomonas aeruginosa) and against fungal pathogens species (Penicillium bilaiae, Aspergillus tamari and Aspergillus sclerotiorum). Likewise, emulsifying properties (emulsification ability (EAI) and stability (ESI) indexes) and the surface characteristics (surface hydrophobicity, ζ-potential and interfacial tension) of the α-La were assessed. Maximum EAI were found at pH 7.0, with higher EAI values for the camel apo α-La (EAI ~19.5 m2/g). This behavior was explained by its relative high surface hydrophobicity and its greater efficiency to reduce the surface tension at the oil-water interface. Furthermore, emulsions were found to be more stable at pH 7.0 compared to pH 5.0 (ESI ~50%) due to the higher electrostatic repulsive forces between oil droplets at pH 7.0 in consistence with the ζ-potential results. This study concluded that the camel apo α-La has antibacterial, antioxidant, and emulsifying properties in agricultural and food industries.

Effects of negative and positive cooperative adsorption of proteins on hydrophobic interaction chromatography media.

The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.

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.

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.

Evaluation of casein as a binding ligand protein for purification of alpha-lactalbumin from beta-lactoglobulin under high hydrostatic pressure.

Fractionation of ß-lactoglubulin (ß-lg) and α-lactalbumin (α-la) using conventional separation technologies remains challenging mainly due to similar molecular weight. Herein, casein (CN) was used as ligand protein to specifically aggregate ß-lg under high hydrostatic pressure (HHP) in order to separate α-la after acidification to pH 4.6. Specifically, we studied the effect of different concentration of CN on α-la purity and recovery. Model solutions of α-la, ß-lg and CN (from 0 to 5 mg/mL) were pressurized (600 MPa-5 min). After acidification and centrifugation of pressure-treated solutions, purity of α-la was increased up to 78% with a recovery of 88% for solution without CN. In contrast with our initial hypothesis, the presence of CN decreased ß-lg pressure-induced aggregation and co-precipitation upon acidification and significantly reduced purity (∼71%). Therefore, our results suggest a chaperone-like activity of CN on ß-lg pressure-induced aggregation which needs further investigation.

One-step chromatographic method to purify α-lactalbumin from whey for nanotube synthesis purposes.

A one-step anion-exchange chromatography method (NaCl gradient elution on a DEAE Sepharose™ Fast Flow gel column) was developed to purify α-lactalbumin (α-LA) from whey protein isolate. α-LA nearly 100% pure (based on the total protein content) was obtained with a yield of about 39%. Besides pure α-LA, which was the main objective of this work, highly pure ß-lactoglobulin was also obtained with a yield of about 59%. The high purity of the obtained α-LA samples allowed its use to synthesise protein nanotubes with excellent gelation properties for their use as food thickeners and bioactive carriers. The samples' purity degree obtained (based on the total protein content) was critical in the formation of proper nanotubes instead of random aggregates, which produced opaque and weak gels, less useful for food applications.

Divergent Anticancer Activity of Free and Formulated Camel Milk α-Lactalbumin.

Alpha-lactalbumin (α-LA), a small milk calcium-binding globular protein, is known to possess noticeable anticancer activity, which is determined by the ability of this protein to form complexes with oleic acid (OA). To date, in addition to human and bovine α-LA, the ability to form such anti-tumor complexes with OA was described for goat and camel α-LA. Although the mechanisms of the anticancer activity of human and bovine α-LA are already well-studied, little is currently known about the anticancer action of this camel protein. The goal of this study was to fill this gap and to analyze the anticancer and pro-apoptotic activities of camel α-LA in its free form (α-cLA) and as an OA-containing complex (OA-α-cLA) using four human cancer cell lines, including Caco-2 colon cancer cells, PC-3 prostate cancer cells, HepG-2 hepatoma cells, and MCF-7 breast cancer cells as targets. The anti-tumor activities of OA-α-cLA and α-cLA were analyzed using MTT test, annexin/PI staining, cell cycle analysis, nuclear staining, and tyrosine kinase (TK) inhibition methods. We show here that the OA-α-cLA complex does not affect normal cells but has noticeable anti-cancer activity, especially against MCF-7 cells, thus boosting the anticancer activity of α-cLA and improving the selectivity of OA. The OA-α-cLA complex mediated cancer cell death via selective induction of apoptosis and cell-cycle arrest at lower IC50 than that of free α-cLA by more than two folds. However, OA induced apoptosis at higher extent than OA-α-cLA and α-cLA. OA also caused unselective apoptosis-dependent cell death in both normal and cancer cells to a similar degree. The apoptosis and cell-cycle arresting effect of OA-α-cLA may be attributed to the TK inhibition activity of OA. Therefore, OA-α-cLA serves as efficient anticancer complex with two functional components, α-cLA and OA, possessing different activities. This study declared the effectiveness of OA-α-cLA complex as a promising entity with anticancer activity, and these formulated OA-camel protein complexes constitute an auspicious approach for cancer remedy, particularly for breast cancer.

Simultaneous quantification of α-lactalbumin and ß-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards.

In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and ß-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and ß-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R(2)>0.99) and recoveries (97.2-102.5% for α-lactalbumin and 99.5-100.3% for ß-casein). The limit of quantification for α-lactalbumin and ß-casein was 8.0mg/100g and 1.2mg/100g, respectively. CVs for α-lactalbumin and ß-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and ß-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5-578.2mg/100g and 116.4-467.4mg/100g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and ß-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids.

Fractionation of sheep cheese whey by a scalable method to sequentially isolate bioactive proteins.

This study reports a procedure for the simultaneous purification of glyco(caseino)macropeptide, immunoglobulin, lactoperoxidase, lactoferrin, α-lactalbumin and ß-lactoglobulin from sheep cheese sweet whey, an under-utilized by-product of cheese manufacture generated by an emerging sheep dairy industry in New Zealand. These proteins have recognized value in the nutrition, biomedical and health-promoting supplements industries. A sequential fractionation procedure using economical anion and cation exchange chromatography on HiTrap resins was evaluated. The whey protein fractionation is performed under mild conditions, requires only the adjustment of pH between ion exchange chromatography steps, does not require buffer exchange and uses minimal amounts of chemicals. The purity of the whey protein fractions generated were analyzed by reversed phase-high performance liquid chromatography and the identity of the proteins was confirmed by mass spectrometry. This scalable procedure demonstrates that several proteins of recognized value can be fractionated in reasonable yield and purity from sheep cheese whey in one streamlined process.

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components.

The HAMLET family of compounds (Human Alpha-lactalbumin Made Lethal to Tumours) was discovered during studies on the properties of human milk, and is a class of protein-lipid complexes having broad spectrum anti-cancer, and some specific anti-bacterial properties. The structure of HAMLET-like compounds consists of an aggregation of partially unfolded protein making up the majority of the compound's mass, with fatty acid molecules bound in the hydrophobic core. This is a novel protein-lipid structure and has only recently been derived by small-angle X-ray scattering analysis. The structure is the basis of a novel cytotoxicity mechanism responsible for anti-cancer activity to all of the around 50 different cancer cell types for which the HAMLET family has been trialled. Multiple cytotoxic mechanisms have been hypothesised for the HAMLET-like compounds, but it is not yet clear which of those are the initiating cytotoxic mechanism(s) and which are subsequent activities triggered by the initiating mechanism(s). In addition to the studies into the structure of these compounds, this review presents the state of knowledge of the anti-cancer aspects of HAMLET-like compounds, the HAMLET-induced cytotoxic activities to cancer and non-cancer cells, and the several prospective cell membrane and intracellular targets of the HAMLET family. The emerging picture is that HAMLET-like compounds initiate their cytotoxic effects on what may be a cancer-specific target in the cell membrane that has yet to be identified. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

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.

A novel ultra-high performance liquid chromatography method for the rapid determination of ß-lactoglobulin as heat load indicator in commercial milk samples.

The level of undenatured acid-soluble ß-lactoglobulin can be used as an indicator to assess the heat load applied to liquid milk, thus further allowing the discrimination between milk originating from different thermal production processes. In this work, a new UHPLC method for the rapid determination of bovine ß-lactoglobulin in 1.8min only (total runtime 3min) is presented using simple UV detection at 205nm. Separation selectivity for possibly co-eluting other major whey proteins (bovine serum albumin, lactoferrin, α-lactalbumin, immunoglobulin G) was verified, and the method validated for the analysis of liquid milk samples regarding linearity (20-560µg/mL, R(2)>0.99), instrumentation precision (RSDs<2.8%), limits of detection and quantification (7 and 23mg/L milk), repeatability of sample work-up (RSDs≤2.6%) and method recovery (103%). In total, 71 commercial liquid milk samples produced using different preservation techniques (e.g., thermal or mechanical treatment), hence featuring different applied heat loads, were profiled for their intrinsic undenatured acid-soluble ß-lactoglobulin levels. As expected, pasteurized milk showed the highest concentrations clearly above 3000mg/L due to pasteurization being the mildest thermal treatment, while in contrast, ultra-high temperature heated milk featured the lowest amounts (<200mg/L). For extended shelf life (ESL) milk, quite diverse levels were determined ranging from ∼100 up to 4000mg/L, thus clearly illustrating variable applied heat loads and impacts on the "nativeness" of milk essentially due to the fact that the production technologies used for ESL milk may differ significantly, and are currently not regulated in the EU.

α-Lactalbumin nanoparticles prepared by desolvation and cross-linking: structure and stability of the assembled protein.

A key step in the preparation of cross-linked protein nanoparticles involves the desolvation of proteins with an organic solvent, which is thought to act by modulating hydrophobic interactions. However, to date, no study has examined the conformational changes that proteins undergo during the assembly process. In this work, by using several biophysical techniques (CD spectroscopy, DSC, TEM, etc.), we studied spheroidal nanoparticles made from bovine α-lactalbumin cross-linked with glutaraldehyde in the presence of acetone. Within the nanoparticle, the polypeptide chain acquires a ß-strand-like conformation (completely different from the native protein in secondary and tertiary structure) in which several side chains likely become available for reacting with glutaraldehyde. A multiplicity of cross-linking sites, together with the polymeric nature of glutaraldehyde, may thus explain the low dry-weight fraction of protein that was found in the nanoparticles. Although covalent bonds undoubtedly constitute the main source for nanoparticle stability, noncovalent interactions also appear to play a role in this regard.

Nanotubular structures developed from whey-based α-lactalbumin fractions for food applications.

Whey proteins have high nutritional value providing use in dietary purposes and improvement of technological properties in processed foods. Functionality of the whey-based α-lactalbumin (α-La) may be increased when assembled in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure α-La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications. For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. α-La and, additionally ß-Lg, were purified at the same batches with the purities in the range of 95%-99%. After enzymatic hydrolysis, WPI-based α-La produced chain-like and long nanotubules with ∼20 nm width while WPC-based α-La produced thinner, miscellaneous, and fibril-like nanostructures by self-assembly. Raman and FT-IR spectroscopies revealed that α-La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy-based α-La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes.

Comparative protein composition analysis of goat milk produced by the Alpine and Saanen breeds in northeastern Brazil and related antibacterial activities.

The protein composition of goat milk differs between goat breeds and could present regional trends. The aim of this study was to comparatively analyze the protein composition of goat milk produced by the Alpine and Saanen breeds in northeastern Brazil and to evaluate the antibacterial activity of its protein fractions. SDS-PAGE, 2-DE electrophoresis and RP-HPLC analyses revealed the absence of αs1-casein in the milk of both breeds and no differences between the αs2-casein, ß-casein, ß-lactoglobulin and α-lactalbumin profiles. The amounts of soluble proteins and ß-casein hydrolysis residues were higher in Saanen milk. Only the protein fraction containing the largest amounts of casein (F60-90%) inhibited bacterial growth, with MIC values between 50 and 100 mg/mL. This study describe for the first time three important points about the goat milk protein of two Brazilian goat breeders: absence of α-s1 casein in the protein profile, differences between the milk protein composition produced by goats of Alpine and Saanen breeders and antibacterial activity of unbroken proteins (casein-rich fraction) present in these milk.

Spermine Sepharose as a clustered-charge anion exchange adsorbent.

We previously showed that the affinity and capacity of ion exchange adsorbents of a given total charge density are improved by immobilization of the charges in pre-ordered clusters, rather than individually in random locations. This previous work used pentalysinamide and pentaargininamide as clustered ligands. This approach allows close control of cluster size, but is uneconomically expensive for some research and most practical applications. In this work, we demonstrate that the inexpensive synthetic analog of the natural polyamine spermine (H2N-CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-NH-CH2-CH2-CH2-NH2) also can serve as the basis of effective clustered adsorbents. The calcium-depleted form of the protein α-lactalbumin, and RNA from baker's yeast, were adsorbed on a spermine Sepharose adsorbent. This adsorbent exhibited enhanced α-lactalbumin binding capacity (Qmax>1.6 and 1.3-fold higher than those for Qiagen DEAE and GE DEAE Sepharose adsorbents of much greater charge density) and higher initial binding affinity (Qmax/KD 2.4 and 2.1-fold higher, respectively). The new spermine-based matrix exhibited a higher value of the Z parameter, suggesting an increased number of apparent interaction sites between the protein and the resin, and functioned well in column mode.