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

SDS-PAGE analysis of rabbit sperm and corresponding Western blotting.

<p>SN, soluble fraction; P, sperm cells; WP, sperm cells after washing three times with buffer. A strong cross-reactivity with a polyclonal antiserum raised against pig SAL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111932#pone.0111932-Marchese1" target="_blank">[19]</a> was observed for a protein migrating at about 23 kDa. Staining was much stronger in the soluble fraction; the weak reactivity observed for the sperm cells disappeared after washing the cells, thus indicating the absence of the protein in this sample.</p

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity

<p>The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.</p

MALDI-TOF-MS analysis of the purified tryptic glycopeptides from rabOBP3 as obtained after HILIC enrichment and nanoLC separation.

<p>Spectra acquired in linear mode of the fractions eluting at 15 and 16 min are reported in panel A and B, respectively; shown are the mono-, bi- and tri-antennary complex-type glycan structures N-linked to Asn44 in peptide (44–50). ▪, N-acetyl-glucosamine; •, mannose; ○, galactose; ◂, fucose; ♦, N-acetyl-neuraminic acid.</p

Binding of 1-NPN (left) and selected ligands (right) to rabOBP3 purified from seminal fluid and delipidated with dichloromethane.

<p>The protein binds the fluorescent probe 1-NPN with a dissociation constant of 3.8 µM (SD 0.9, n = 3). None of the ligands tested exhibited strong affinity to the protein, except quercetin, for which a physiological role does not seem plausible. Calculated dissociation constants are 2.2, 7.8 and 11.2 µM for quercetin, 2-nonenal and geraniol, respectively.</p

Three-dimensional model of rabOBP3 as built by using the crystallographic structure of pig SAL (Boar salivary lipocalin, PDB ID: 1 GM6) as a template[10].

<p>Molecular model of rabOBP3 and pig SAL are shown in the left- and right-top panel, respectively. The corresponding sequence alignment is shown in the bottom panel, where conserved amino acids are highlighted in yellow. The conserved N-glycosylation site (Asn44), and oxidized (Cys59 and Cys152) and reduced (Cys66 and Cys133) residues are indicated by specific labelling (top) or asterisks (bottom).</p

Expression of rabOBP3 in different tissues of male (m) and female (f) rabbit individuals.

<p>SDS-PAGE analysis of different rabbit tissues and corresponding Western blotting are shown. M: molecular weight markers; m1: nasal respiratory tissue; m2: epidydimis; m3: testis; m4: prostate; f1: nasal respiratory tissue; f2: uterine tubes; f3: ovaries; f4: uterus; P: purified rabOBP3.</p

MALDI-TOF-MS analysis of the tryptic digest of rabOBP3 alkylated with iodoacetamide under denaturing, non-reducing conditions before (top) and following (bottom) treatment with dithiothreitol.

<p>Constant and variable signals are labelled in the spectra acquired in reflectron mode to highlight reduced and oxidized residues present under native conditions. Trypsin-derived peptides are indicated with an asterisk.</p