A New Method for Growth Factor Enrichment from Dairy Products by Electrodialysis with Filtration Membranes: The Major Impact of Raw Product Pretreatment.

This study is focused on fractionation of insulin-like growth factor I (IGF-I) and transforming growth factor-ß2 (TGF-ß2) using a new electro-based membrane process calledelectrodialysis with filtration membranes (EDFM). Before EDFM, different pretreatments were tested, and four pH conditions (4.25, 3.85, 3.45, and 3.05) were used during EDFM. It was demonstrated that a 1:1 dilution of defatted colostrum with deionized water to decrease mineral content followed by the preconcentration of GFs by UF is necessary and allow for these compounds to migrate to the recovery compartment during EDFM. MS analyses confirmed the migration, in low quantity, of only α-lactalbumin (α-la) and ß-lactoglobulin (ß-lg) from serocolostrum to the recovery compartment during EDFM. Consequently, the ratio of GFs to total protein in recovery compartment compared to that of feed serocolostrum solution was 60× higher at pH value 3.05, the optimal pH favoring the migration of IGF-I and TGF-ß2. Finally, these optimal conditions were tested on acid whey to also demonstrate the feasibility of the proposed process on one of the main by-products of the cheese industry; the ratio of GFs to total protein was 2.7× higher in recovery compartment than in feed acid whey solution, and only α-la migrated. The technology of GF enrichment for different dairy solutions by combining ultrafiltration and electrodialysis technologies was proposed for the first time.

The Effect of Hyperbaric Storage on the Nutritional Value and Retention of Certain Bioactive Proteins in Human Milk.

Human milk (HM) contains the essential macronutrients and bioactive compounds necessary for the normal growth and development of newborns. The milk collected by human milk banks is stored frozen and pasteurized, reducing its nutritional and biological value. The purpose of this study was to determine the effect of hyperbaric storage at subzero temperatures (HS-ST) on the macronutrients and bioactive proteins in HM. As control samples, HM was stored at the same temperatures under 0.1 MPa. A Miris HM analyzer was used to determine the macronutrients and the energy value. The lactoferrin (LF), lysozyme (LYZ) and α-lactalbumin (α-LAC) content was checked using high-performance liquid chromatography, and an ELISA test was used to quantify secretory immunoglobulin A (sIgA). The results showed that the macronutrient content did not change significantly after 90 days of storage at 60 MPa/-5 °C, 78 MPa/-7 °C, 111 MPa/-10 °C or 130 MPa/-12 °C. Retention higher than 90% of LYZ, α-LAC, LF and sIgA was observed in the HM stored at conditions of up to 111 MPa/-10 °C. However, at 130 MPa/-12 °C, there was a reduction in LYZ and LF, by 39 and 89%, respectively. The storage of HM at subzero temperatures at 0.1 MPa did not affect the content of carbohydrates or crude and true protein. For fat and the energy value, significant decreases were observed at -5 °C after 90 days of storage.

A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome.

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

The effect of heat treatment on the lactosylation of milk proteins.

Protein lactosylation is a significant modification that occurs during the heat treatment of dairy products, causing changes in proteins' physical-chemical and nutritional properties. Knowledge of the detailed lactosylation information on milk proteins under various heat treatments is important for selecting appropriate thermo-processing and identifying markers to monitor heat load in dairy products. In the present study, we used proteomics techniques to investigate lactosylated proteins under different heating temperatures. We observed a total of 123 lactosylated lysines in 65 proteins, with lactosylation even occurring in raw milk. The number of lactosylated lysines and proteins increased moderately at 75°C to 130°C, but dramatically at 140°C. We found that 6 out of 10, 9 out of 16, 6 out of 12, and 5 out of 15 lysine residues in κ-casein, ß-lactoglobulin, α-lactalbumin, and αS1-casein, respectively, were lactosylated under the applied heating treatment. Moreover, different lactosylation states of individual lysines and proteins can indicate the intensity of heating processes. Lactosylation of K14 in ß-lactoglobulin could distinguish pasteurized and UHT milk, while lactosylation of lactotransferrin can reflect moderate heat treatment of products.

Exclusive Human Milk Diet for Extremely Premature Infants: A Novel Fortification Strategy That Enhances the Bioactive Properties of Fresh, Frozen, and Pasteurized Milk Specimens.

Background: Human milk (HM) fortification has been recommended for the nutritional optimization of very low-birthweight infants. This study analyzed the bioactive components of HM and evaluated fortification choices that could accentuate or attenuate the concentration of such components, with special reference to human milk-derived fortifier (HMDF) offered to extremely premature infants as an exclusive human milk diet. Materials and Methods: An observational feasibility study analyzed the biochemical and immunochemical characteristics of mothers' own milk (MOM), both fresh and frozen, and pasteurized banked donor human milk (DHM), each supplemented with either HMDF or cow's milk-derived fortifier (CMDF). Gestation-specific specimens were analyzed for macronutrients, pH, total solids, antioxidant activity (AA), α-lactalbumin, lactoferrin, lysozyme, and α- and ß-caseins. Data were analyzed for variance applying general linear model and Tukey's test for pairwise comparison. Results: DHM exhibited significantly lower (p < 0.05) lactoferrin and α-lactalbumin concentrations than fresh and frozen MOM. HMDF reinstated lactoferrin and α-lactalbumin and exhibited higher protein, fat, and total solids (p < 0.05) in comparison to unfortified and CMDF-supplemented specimens. HMDF had the highest (p < 0.05) AA, suggesting the potential capability of HMDF to enhance oxidative scavenging. Conclusion: DHM, compared with MOM, has reduced bioactive properties, and CMDF conferred the least additional bioactive components. Reinstatement and further enhancement of bioactivity, which has been attenuated through pasteurization of DHM, is demonstrated through HMDF supplementation. Freshly expressed MOM fortified with HMDF and given early, enterally, and exclusively (3E) appears an optimal nutritional choice for extremely premature infants.

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro.

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including ß-lactoglobulin (ß-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92-100, 125-135, 125-138, and 149-162) in ß-LG, 2 peptides in α-LA (AA 80-93 and 63-79), 2 peptides in αS1-casein (AA 84-90 and 125-132), and 1 peptide (AA 25-32) in αS2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.

Effects of a Novel High-Quality Protein Infant Formula on Energetic Efficiency and Tolerance: A Randomized Trial.

OBJECTIVES: Protein overfeeding in infants can have negative effects, such as diabetes and childhood obesity; key to reducing protein intake from formula is improving protein quality. The impact of a new infant formula [study formula (SF)] containing alpha-lactalbumin, lactoferrin, partially hydrolyzed whey, and whole milk on growth and tolerance compared to a commercial formula (CF) and a human milk reference arm was evaluated. METHODS: This randomized, double-blind trial included healthy, singleton, term infants, enrollment age ≤14 days. Primary outcome was mean daily weight gain. Secondary outcomes were anthropometrics, formula intake, serum amino acids, adverse events, gastrointestinal characteristics, and general disposition. RESULTS: Non-inferiority was demonstrated. There were no differences between the formula groups for z scores over time. Formula intake [-0.33 oz/kg/day, 95% confidence interval (CI): -0.66 to -0.01, P = 0.05] and mean protein intake (-0.13 g/kg/day, 95% CI: -0.26 to 0.00, P = 0.05) were lower in the SF infants, with higher serum essential amino acid concentrations (including tryptophan) compared to the CF infants. Energetic efficiency was 14.0% (95% CI: 8.3%, 19.7%), 13.0% (95% CI: 6.0%, 20.0%), and 18.1% (95% CI: 9.4%, 26.8%) higher for weight, length, and head circumference, respectively, in SF infants compared to the CF infants. SF infants had significantly fewer spit-ups and softer stool consistency than CF infants. CONCLUSIONS: The SF resulted in improved parent-reported gastrointestinal tolerance and more efficient growth with less daily formula and protein intake supporting that this novel formula may potentially reduce the metabolic burden of protein overfeeding associated with infant formula.

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.

Protein Quality in Infant Formulas Marketed in Brazil: Assessments on Biodigestibility, Essential Amino Acid Content and Proteins of Biological Importance.

Infant formulas, designed to provide similar nutritional composition and performance to human milk, are recommended when breastfeeding is not enough to provide for the nutritional needs of children under 12 months of age. In this context, the present study aimed to assess the protein quality and essential amino acid content of both starting (phase 1) and follow-up (phase 2) formulas from different manufacturers. The chemical amino acid score and protein digestibility corrected by the amino acid score were calculated. The determined protein contents in most formulas were above the maximum limit recommended by FAO and WHO guidelines and at odds with the protein contents declared in the label. All infant formulas contained lactoferrin (0.06 to 0.44 g·100 g-1) and α-lactalbumin (0.02 to 1.34 g·100 g-1) below recommended concentrations, whereas ĸ-casein (8.28 to 12.91 g·100 g-1), α-casein (0.70 to 2.28 g·100 g-1) and ß-lactoglobulin (1.32 to 4.19 g·100 g-1) were detected above recommended concentrations. Essential amino acid quantification indicated that threonine, leucine and phenylalanine were the most abundant amino acids found in the investigated infant formulas. In conclusion, infant formulas are still unconforming to nutritional breast milk quality and must be improved in order to follow current global health authority guidelines.

Benzyl isothiocyanate-modified α-lactalbumin - Two-dimensional high-performance thin-layer chromatography for analyzing modified peptides.

In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae - benzyl isothiocyanate (BITC) - and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.

Analysis and comparison of key proteins in Maiwa yak and bovine milk using high-performance liquid chromatography mass spectrometry.

Yak milk is an essential and predominant food resource for Tibetan people for subsistence purposes and to combat altitude-induced challenges. Due to its unique qualities, yak milk has recently been gaining broader attention from consumers across China as well in other parts of the world. One of the key characteristics of yak milk is the protein content, which is about 40 to 60% higher than that of native bovine milk. In this work, a sensitive and reproducible high-throughput analytical method was developed employing both ultra high-performance liquid chromatography Orbitrap (Thermo Fisher Scientific) high-resolution accurate mass spectroscopy (UHPLC-HRAM-MS) and UHPLC coupled with triple quadrupole tandem MS (UHPLC-QqQ-MS) to simultaneously analyze 8 milk proteins. A total of 15 Maiwa yak milk samples and 15 bovine milk samples were qualitatively and quantitatively analyzed using targeted proteomics and compared for α-lactalbumin, ß-lactoglobulin, αS1-casein, αS2-casein, ß-casein, κ-casein, lactoferrin, and osteopontin. Peptides of ß-lactoglobulin were used to specifically distinguish yak and bovine milk. The results showed that this novel detection method could quantitatively detect these major and minor milk proteins with >0.99 linear correlation coefficient and a recovery rate between 90 and 120%, with relative standard deviations typically less than 10%. The data revealed that yak milk not only had higher overall milk protein content than bovine milk but higher lactoferrin and osteopontin contents as well. The lactoferrin content of yak milk was about 30% higher than that of bovine milk, and the osteopontin content of yak milk was nearly twice that of bovine milk. The application of this method demonstrates that UHPLC-HRAM-MS and UHPLC-QqQ-MS are suitable for high-throughput qualitative and quantitative analysis of major and minor proteins of yak and bovine milk.

Immunological Analysis of Isothiocyanate-Modified α-Lactalbumin Using High-Performance Thin Layer Chromatography.

Undirected modifications between food proteins and secondary plant metabolites can occur during food processing. The results of covalent interactions can alter the functional and biological properties of the proteins. The present work studied the extent of which covalent conjugation of the bioactive metabolite benzyl isothiocyanate (BITC; a glucosinolate breakdown product) to the whey protein α-lactalbumin affects the protein's allergenicity. Additional to the immunological analysis of native untreated and BITC-modified α-lactalbumin, the analysis of antigenic properties of proteolytically digested protein derivatives was also performed by high performance thin layer chromatography and immunostaining. As a result of the chemical modifications, structural changes in the protein molecule affected the allergenic properties. In this process, epitopes are destroyed or inactivated, but at the same time, buried epitopes can be exposed or newly formed, so that the net effect was an increase in allergenicity, in this case. Results from the tryptic hydrolysis suggest that BITC conjugation sterically hindered the cleavage sites for the enzyme, resulting in reduced digestibility and allergenicity. Residual antigenicity can be still present as short peptide fragments that provide epitopes. The desire to make food safer for allergy sufferers and to protect sensitized individuals from an allergenic reaction makes it clear that the detection of food antigens is mandatory; especially by considering protein interactions.

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.

Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins.

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, ß-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% - 103%) and recovery (96% - 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.

Short communication: Caseins and α-lactalbumin content of camel milk (Camelus dromedarius) determined by capillary electrophoresis.

Camel milk has unique physical, nutritional, and technological properties when compared with other milks, especially bovine. Because proteins confer many of the properties of milk and its products, this study aimed to determine the proteins of camel milk, their correlations, and relative distribution. Raw milk samples were collected from 103 dromedary camels in the morning and evening. Capillary electrophoresis results showed wide variation in the concentrations (g/L) of proteins between samples as follows: α-lactalbumin, 0.3 to 2.9; αS1-casein, 2.4 to 10.3; αS2-casein, 0.3 to 3.9; ß-casein, 5.5 to 29.0; κ-casein, 0.1 to 2.4; unknown casein protein 1, 0.0 to 3.4; and unknown casein protein 2, 0.0 to 4.6. The range in percent composition of the 4 caseins were as follows: αS1, 12.7 to 35.3; αS2, 1.8 to 20.8; ß, 42.3 to 77.4; and κ, 0.6 to 17.4. The relative proportion of αS1-, αS2-, ß-, and κ-caseins in camel milk (26:4:67:3, wt/wt) differed from that of bovine milk (38:10:36:12, wt/wt). This difference might explain the dissimilarity between the 2 milks with respect to technical and nutritional properties.

Front-face fluorescence spectroscopy combined with chemometrics to detect high proteinaceous matter in milk and whey ultrafiltration permeate.

Proteinaceous matter can leak into the permeate stream during ultrafiltration (UF) of milk and whey and lead to financial losses. Although manufacturers can measure protein content in the finished permeate powders, there is currently no rapid monitoring tool during UF to identify protein leak. This study applied front-face fluorescence spectroscopy (FFFS) and chemometrics to identify the fluorophore of interest associated with the protein leak, develop predictive models to quantify true protein content, and classify the types of protein leak in permeate streams. Crude protein (CP), nonprotein nitrogen (NPN), true protein (TP), tryptone-equivalent peptide (TEP), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) contents were measured for 37 lots of whey permeate and 29 lots of milk permeate from commercial manufacturers. Whey permeate contained more TEP than did milk permeate, whereas milk permeate contained more α-LA and ß-LG than did whey permeate. The types of protein leak were thus identified for predictive model development. Based on excitation-emission matrix (EEM) of high- and low-TP permeates, tryptophan excitation spectra were collected for predictive model development, measuring TP content in permeate. With external validation, a useful model for quality control purposes was developed, with a root mean square error of prediction of 0.22% (dry basis) and a residual prediction deviation of 2.8. Moreover, classification models were developed using partial least square discriminant analysis. These classification methods can detect high TP level, high TEP level, and presence of α-LA or ß-LG with 83.3%, 84.8%, and 98.5% cross-validated accuracy, respectively. This method showed that FFFS and chemometrics can rapidly detect protein leaks and identify the types of protein leak in UF permeate. Implementation of this method in UF processing plants can reduce financial loss from protein leaks and maintain high-quality permeate production.

Synthesis and characterization of a new MeCAT reagent containing a photocleavable linker for labeling of proteins and peptides in mass spectrometric analyses.

The quantification of proteins and peptides becomes more important besides mere identification in modern life sciences. Therefore, we have developed a new reagent that adds to the known metall-coded affinity tagging strategy employed in molecular and elemental mass spectrometry containing a photocleavable linker. A synthesis route was developed that provides the new reagent in good yields. The stability of the synthesized reagents was assessed under different temperature and illumination conditions. Labeling reactions were carried out at peptide and protein level, while also the fragmentation behavior of labeled peptides was assessed. In additional experiments, the photocleavability of the new reagent was examined. Upon irradiation with ultraviolet light, the photoproducts were liberated and could be used for quantification of labeled peptides.

Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS).

Hydrogen deuterium exchange mass spectrometry (HDX-MS) has significant potential for protein structure initiatives but its relationship with protein conformations is unclear. We report on the efficacy of HDX-MS to distinguish between native and non-native proteins using a popular approach to calculate HDX protection factors (PFs) from protein structures. The ability of HDX-MS to identify native protein conformations is quantified by binary structural classification such that merits of the approach for protein modelling can be quantified and better understood. We show that highly accurate PF calculations are not a prerequisite for HDX-MS simulations that are capable of effectively discriminating between native and non-native protein folds. The simulations can also be performed directly on unique structures facilitating high-throughput evaluation of many alternate conformations. The ability of HDX-MS to classify the conformations of homo-protein assemblies is also investigated. In contrast to protein monomers, we show a significant lack of correspondence between the simulated and experimental HDX-MS data for these systems with a subsequent decrease in the ability of HDX-MS to identify native states. However, we demonstrate surprisingly high diagnostic ability of the simulated data for assemblies in which a significant proportion of the individual chains occupy protein-protein interfaces. We relate this to the number of peptides that can sample alternate subunit orientations and discuss these observations within the larger context of applying HDX-MS to evaluate protein structures. Graphical Abstract.

Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.

Electrochemical immunoassay for lactalbumin based on the use of ferrocene-modified gold nanoparticles and lysozyme-modified magnetic beads.

The authors describe a method for electrochemical determination of the breast cancer biomarker α-lactalbumin (α-LA) using disposable screen-printed carbon electrodes (SPCEs). Lysozyme-conjugated Fe3O4 nanoparticles (Lys-Fe3O4NPs) were used to capture α-LA on the surface of the SPCEs which then is trapped in an immunosandwich using secondary antibodies labeled with ferrocene-modified gold nanoparticles. The amperometric response of ferrocene (recorded at +0.1 V vs. silver pseudo-reference electrode) as well as the electrocatalytic activity of gold nanoparticles on the hydrogen evolution reaction (recorded at -1.0 V Vs Ag pseudo-reference electrode) was exploited to sense α-LA. A sensitive voltammetric response is observed, with (a) a sensitivity of 0.8789 µA·nM-1.cm-2, (b) a detection limit (LOD, at S/N = 3) as low as 0.07 ng·mL-1, and (c) linear response in the 0.75 to 630 ng mL-1 α-LA concentration range. The assay is selective and reproducible, and the SPCEs have good storage stability. The SPCEs were applied (a) to the analysis of (spiked) maternal milk, (b) of spiked serum from healthy and pregnant persons, and (c) of serum of patients suffering from breast cancer. Graphical abstract Schematic presentation of a sensitive electrochemical immunoassay platform based on ferrocene modified gold nanoparticles and lysozyme modified magnetic beads for the determination of alpha lactalbumin in human sera and breast milk by the amperometric response of ferrocene and hydrogen evolution reaction.