An Extensive Description of the Peptidomic Repertoire of the Hen Egg Yolk Plasma

Hen egg is a raw material widely used for the preparation of food,  pharmaceutical and cosmetoceutical products. Dedicated proteomic studies were accomplished on eggshell membrane, egg white, and yolk, identifying the most abundant proteins. No similar peptidomic studies have been performed so far. Only preliminary investigations on bioactive peptides in egg fractions and digestates were accomplished through functional screening assays, characterizing antioxidant, antibacterial, antiviral, immunomodulatory, and antihypertensive preparations and isolated components. This study fills this gap and provides a comprehensive picture of the peptides present in the yolk plasma of different hen egg types after 24 and 264 h of laying, taking advantage of a procedure based on a two-step fractionation followed by combined MALDI-TOF-TOF-MS- and nanoLC-ESI-Q-Orbitrap-MS/MS-based analysis. Six hundred and twenty-eight peptides were characterized as deriving from the proteolytic processing of larger protein components after the physiological action of chicken chymotrypsin-like and pepsin-like enzymes. Structural details on their post-translational modifications were also provided. Identified peptides were subjected to bioinformatic analysis and further compared with available data from the literature, ascertaining 198 peptides associable with putative antihypertensive, antimicrobial, anticancer, antiviral, antibiofilm, anorectic, calcium-binding, and anti-inflammatory activities. This analysis was often confirmative of previous experimental evidence on functional properties of unfractionated preparations or isolated molecules. These results further emphasize the bioactive action of yolk-derived peptides as related to egg consumption, and the potential use of these molecules as additive ingredients in the preparation of functional foods and cosmetics

An Extensive Description of the Peptidomic Repertoire of the Hen Egg Yolk Plasma

Hen egg is a raw material widely used for the preparation of food,  pharmaceutical and cosmetoceutical products. Dedicated proteomic studies were accomplished on eggshell membrane, egg white, and yolk, identifying the most abundant proteins. No similar peptidomic studies have been performed so far. Only preliminary investigations on bioactive peptides in egg fractions and digestates were accomplished through functional screening assays, characterizing antioxidant, antibacterial, antiviral, immunomodulatory, and antihypertensive preparations and isolated components. This study fills this gap and provides a comprehensive picture of the peptides present in the yolk plasma of different hen egg types after 24 and 264 h of laying, taking advantage of a procedure based on a two-step fractionation followed by combined MALDI-TOF-TOF-MS- and nanoLC-ESI-Q-Orbitrap-MS/MS-based analysis. Six hundred and twenty-eight peptides were characterized as deriving from the proteolytic processing of larger protein components after the physiological action of chicken chymotrypsin-like and pepsin-like enzymes. Structural details on their post-translational modifications were also provided. Identified peptides were subjected to bioinformatic analysis and further compared with available data from the literature, ascertaining 198 peptides associable with putative antihypertensive, antimicrobial, anticancer, antiviral, antibiofilm, anorectic, calcium-binding, and anti-inflammatory activities. This analysis was often confirmative of previous experimental evidence on functional properties of unfractionated preparations or isolated molecules. These results further emphasize the bioactive action of yolk-derived peptides as related to egg consumption, and the potential use of these molecules as additive ingredients in the preparation of functional foods and cosmetics

MALDI-TOF-MS Platform for Integrated Proteomic and Peptidomic Profiling of Milk Samples Allows Rapid Detection of Food Adulterations

Adulteration of ovine, caprine, and buffalo milks with more common bovine material occurs for economic reasons and seasonal availability. Frauds are also associated with the use of powdered milk instead of declared, fresh material. In this context, various analytical methods have been adapted to dairy science applications with the aim to evaluate adulteration of milk samples, although time-consuming, suitable only for speciation or thermal treatment analysis, or useful for a specific fraud type. An integrated MALDI-TOF-MS platform for the combined peptidomic and proteomic profiling of milk samples is here presented, which allows rapid detection of illegal adulterations due to the addition of either nondeclared bovine material to water buffalo, goat, and ovine milks or of powdered bovine milk to the fresh counterpart. Peptide and protein markers of each animal milk were identified after direct analysis of a large number of diluted skimmed and/or enriched diluted skimmed filtrate samples. In parallel, markers of thermal treatment were characterized in different types of commercial milks. Principal components scores of ad hoc prepared species- or thermal treatment-associated adulterated milk samples were subjected to partial least-squares regression, permitting a fast accurate estimate of the fraud extents in test samples at either protein and peptide level. With respect to previous reports on MALDI-TOF-MS protein profiling methodologies for milk speciation, this study extends that approach to the analysis of the thermal treatment and introduces an independent, complementary peptide profiling measurement, which integrates protein data with additional information on peptides, validating final results and ultimately broadening the method applicability

Human Milk Proteins: An Interactomics and Updated Functional Overview

Milk and milk fractions are characterized by a wide array of proteins, whose concentration spans across several orders of magnitude. By exploiting a combined approach based on functional gene ontology enrichment (FatiGO/Babelomics), hierarchical clustering, and pathway and network analyses, we merged data from literature dealing with protein-oriented studies on human milk. A total of 285 entries defined a nonredundant list upon comparison with the Ingenuity Knowledge Base from the Ingenuity Pathway Analysis software. Results were compared with an inventory of bovine milk proteins gathered from dedicated proteomic studies. A protein core of 106 proteins was found, with most of the entries associated to three main biological functions, namely nutrient transport/lipid metabolism, concretization of the immune system response and cellular proliferation processes. Our analyses confirm and emphasize that the biological role of the human milk proteins is not only limited to the provision of external nutrients and defense molecules against pathogens to the suckling but also to the direct stimulation of the growth of neonate tissues/organs and to the development of a proper independent immune system, both through the induction of a number of molecular cascades associated with cell proliferation/differentiation. The latter aspects were previously investigated by single-molecule dedicated studies, missing the holistic view that results from our analysis

MALDI-TOF-MS Platform for Integrated Proteomic and Peptidomic Profiling of Milk Samples Allows Rapid Detection of Food Adulterations

Adulteration of ovine, caprine, and buffalo milks with more common bovine material occurs for economic reasons and seasonal availability. Frauds are also associated with the use of powdered milk instead of declared, fresh material. In this context, various analytical methods have been adapted to dairy science applications with the aim to evaluate adulteration of milk samples, although time-consuming, suitable only for speciation or thermal treatment analysis, or useful for a specific fraud type. An integrated MALDI-TOF-MS platform for the combined peptidomic and proteomic profiling of milk samples is here presented, which allows rapid detection of illegal adulterations due to the addition of either nondeclared bovine material to water buffalo, goat, and ovine milks or of powdered bovine milk to the fresh counterpart. Peptide and protein markers of each animal milk were identified after direct analysis of a large number of diluted skimmed and/or enriched diluted skimmed filtrate samples. In parallel, markers of thermal treatment were characterized in different types of commercial milks. Principal components scores of ad hoc prepared species- or thermal treatment-associated adulterated milk samples were subjected to partial least-squares regression, permitting a fast accurate estimate of the fraud extents in test samples at either protein and peptide level. With respect to previous reports on MALDI-TOF-MS protein profiling methodologies for milk speciation, this study extends that approach to the analysis of the thermal treatment and introduces an independent, complementary peptide profiling measurement, which integrates protein data with additional information on peptides, validating final results and ultimately broadening the method applicability

Human Milk Proteins: An Interactomics and Updated Functional Overview

Milk and milk fractions are characterized by a wide array of proteins, whose concentration spans across several orders of magnitude. By exploiting a combined approach based on functional gene ontology enrichment (FatiGO/Babelomics), hierarchical clustering, and pathway and network analyses, we merged data from literature dealing with protein-oriented studies on human milk. A total of 285 entries defined a nonredundant list upon comparison with the Ingenuity Knowledge Base from the Ingenuity Pathway Analysis software. Results were compared with an inventory of bovine milk proteins gathered from dedicated proteomic studies. A protein core of 106 proteins was found, with most of the entries associated to three main biological functions, namely nutrient transport/lipid metabolism, concretization of the immune system response and cellular proliferation processes. Our analyses confirm and emphasize that the biological role of the human milk proteins is not only limited to the provision of external nutrients and defense molecules against pathogens to the suckling but also to the direct stimulation of the growth of neonate tissues/organs and to the development of a proper independent immune system, both through the induction of a number of molecular cascades associated with cell proliferation/differentiation. The latter aspects were previously investigated by single-molecule dedicated studies, missing the holistic view that results from our analysis

Human Milk Proteins: An Interactomics and Updated Functional Overview

Milk and milk fractions are characterized by a wide array of proteins, whose concentration spans across several orders of magnitude. By exploiting a combined approach based on functional gene ontology enrichment (FatiGO/Babelomics), hierarchical clustering, and pathway and network analyses, we merged data from literature dealing with protein-oriented studies on human milk. A total of 285 entries defined a nonredundant list upon comparison with the Ingenuity Knowledge Base from the Ingenuity Pathway Analysis software. Results were compared with an inventory of bovine milk proteins gathered from dedicated proteomic studies. A protein core of 106 proteins was found, with most of the entries associated to three main biological functions, namely nutrient transport/lipid metabolism, concretization of the immune system response and cellular proliferation processes. Our analyses confirm and emphasize that the biological role of the human milk proteins is not only limited to the provision of external nutrients and defense molecules against pathogens to the suckling but also to the direct stimulation of the growth of neonate tissues/organs and to the development of a proper independent immune system, both through the induction of a number of molecular cascades associated with cell proliferation/differentiation. The latter aspects were previously investigated by single-molecule dedicated studies, missing the holistic view that results from our analysis

Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples

To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S–S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S–S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S–S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity

Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples

To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S–S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S–S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S–S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity

Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples

To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S–S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S–S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S–S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity