Food proteins and peptides

The qualitative and quantitative determination of proteins and peptides in raw or processed food is experiencing a growing interest and importance from both scientific and economic point of view. Proteomics and peptidomics are relatively new entries in the field of food security, safety and authenticity, and themselves can contribute to the emergence of new branches of the science of food, such as foodomics and the just born nutriomics, digestomics, and gut metagenomics/metaproteomics. Mass spectrometry, in combination with a wide variety of separation methods and bioinformatic tools, is the principal methodology for proteomics. Both the so-called "in-gel" and "gel-free shotgun" bottom-up approaches are widely used.Among the arguments described in this chapter there are: stress effects on gene expression, postharvest (plant) and postmortem (livestock) protein modification, food safety, quality and authentication, food processing and quality control, frauds discovery, food peptidomics and digestomics. © 2015 Elsevier B.V

Obtaining Of β-Lactoglobulin By Gel Filtration Of Cow Milk Whey

Milk whey proteins carry out a number of important biological functions and also they are precursors of many biologically active peptides (antihypertensive peptides, antagonists of opioid receptors, regulators of intestinal motility, immunomodulatory, anti-microbial and anti-cancer peptides, appetite regulators and so on.). An important stage in natural bioactive peptides obtaining from milk whey proteins is the isolation of homogeneous proteins-precursors. Considering the significant difference in the molecular masses of whey proteins, a promising method for their selection is gel filtration. The purpose of the research was the fractionation of bioactive peptides precursors from milk whey using gel filtration on Sephadex G-150. The whey was obtained from fresh skimmed milk after isoelectric precipitation of casein. Gel filtration was carried out on the columns from a liquid chromatography kit by the “Reanal” company. The fractional composition and the degree of homogeneity of milk whey proteins were determined by disc-electrophoresis in the plates of a polyacrylamide gel. A repeated gel filtration of fractions from the chromatographic peaks, separated into sections, was performed to increase the fractionation efficiency. While choosing a dextran gel for gel filtration of precursors of biologically active peptides from milk whey proteins, we have taken into account the range of their molecular weights (from 10000 to 150000 Da), the ability to form supramolecular structures (β-LG), as well as the previously obtained results of gel filtration. As a result, it was shown that repeated gel filtration of milk whey on Sephadex G-150 allows efficiently fractionate the proteins-precursors of bioactive peptides. The range of peptides and proteins molecular weights that can be fractionated on this Sephadex is from 5000 to 300 000 Da. The usage of repeated gel filtration on Sephadex G-150 with the chromatogram separation into sectors allows to effectively fractionate proteins-precursors of bioactive peptides from milk whey. In particular, homogeneous β-lactoglobulin (degree of homogeneity > 95 %) and partially purified α-lactalbumin, as well as a group of immunoglobulins and a proteose-peptone fraction were obtained

A nested mixture model for protein identification using mass spectrometry

Mass spectrometry provides a high-throughput way to identify proteins in biological samples. In a typical experiment, proteins in a sample are first broken into their constituent peptides. The resulting mixture of peptides is then subjected to mass spectrometry, which generates thousands of spectra, each characteristic of its generating peptide. Here we consider the problem of inferring, from these spectra, which proteins and peptides are present in the sample. We develop a statistical approach to the problem, based on a nested mixture model. In contrast to commonly used two-stage approaches, this model provides a one-stage solution that simultaneously identifies which proteins are present, and which peptides are correctly identified. In this way our model incorporates the evidence feedback between proteins and their constituent peptides. Using simulated data and a yeast data set, we compare and contrast our method with existing widely used approaches (PeptideProphet/ProteinProphet) and with a recently published new approach, HSM. For peptide identification, our single-stage approach yields consistently more accurate results. For protein identification the methods have similar accuracy in most settings, although we exhibit some scenarios in which the existing methods perform poorly.Comment: Published in at http://dx.doi.org/10.1214/09-AOAS316 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Probing protein sequences as sources for encrypted antimicrobial peptides

Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources

An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins

Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.

Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions

Structural analysis of the variable major proteins of Borrelia hermsii.

Borrelia hermsii undergoes spontaneous antigenic variation in vivo and in vitro. Serotype specificity is associated with expression of one of a family of molecular weight-variable proteins, the pI proteins. We studied the structure of the pI proteins as well as the molecular weight-invariable pII proteins of three serotypes of B. hermsii HS1: C, 7, and 21. The techniques used were one-dimensional (1-D) mapping of Staphylococcus aureus V8 protease-generated peptides and two-dimensional (2-D) mapping of alpha-chymotrypsin-generated peptides. The pI and pII proteins were isolated by excision of polypeptides from stained polyacrylamide gel electropherograms. The 1-D peptide patterns were visualized by fluorography of intrinsically [14C]leucine-labeled proteins or by silver stain. Before 2-D mapping, polypeptides in excised gel fragments were labeled with 125I in the presence of chloramine-T. We also compared the 2-D peptide maps of pI proteins, pI7 and pI21, after their surface-exposed portions were radioiodinated using 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril (Iodogen). The I-D and 2-D peptide maps demonstrated the following: (a) pI proteins of the three serotypes have few V8 protease- or chymotrypsin-generated peptides in common, and (b) pI proteins of each serotype appear to be identical. The findings suggest that pI protein variability derives from extensive differences in the amino acid sequences of these proteins

Chromatographic separation and identification of some peptides in partial hydroylsates of gelatin

Recently we have been engaged in a study of the chemical structure of collagen and gelatin with the object of determining the sequence of the amino acid residues in the polypeptide chains of these proteins. In the course of this study we have made considerable progress in the chromatographic analysis of complex mixtures of peptides and we have isolated and identified several simple peptides which occur in partial hydrolysates of gelatin. The initial separation of the mixture into zones of one or more peptides has been made on a column of ion exchange resin; further separation of the peptides in each zone has been achieved by chromatographing in the form of dinitrophenyl (DNP) peptides on columns of silicic acid-Celite. It is to be hoped that the particular combination of chromatographic methods which has been successfully used in the present study will be helpful in the resolution of the complex mixtures which result from the partial hydrolysis of other proteins

Biodegradable multiblock copolymers for drug delivery applications

With rapid advances in genomic research and biotechnology, an increasing number of pharmaceutical proteins and peptides become available for a variety of diseases. However, the efficient delivery of these drugs is hampered by their large size and (biological) instability. Consequently, to obtain a desired therapeutic effect, proteins and peptides are usually administered parenterally by repeated subcutaneous or intramuscular injection. To overcome the problem of patient compliance, extensive research is performed on the development of controlled release systems

Gastrointestinal endogenous proteins as a source of bioactive peptides : Doctor of Philosophy in Nutritional Sciences at Riddet Institute, Massey University, Palmerston North, New Zealand

Gastrointestinal endogenous proteins (GEP) were investigated as a source of bioactive peptides. In silico and in vitro methods were used singly or in combination to study GEP-derived peptides after simulated digestion. The presence of bioactive peptides after in vivo digestion was determined using a porcine model. Bioactivity of the peptides was assessed using selected in vitro bioactivity assays, and peptides were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In the in silico study, twenty six different GEP and seven dietary proteins were subjected to simulated in silico gastrointestinal (SIGIT) digestion. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified by screening them against an online database of bioactive peptides (BIOPEP). The predicted number of bioactive peptides released after the SIGIT digestion of GEP ranged from 1 (secretin) to 39 (mucin-5AC), while those for dietary proteins ranged from 1 (gliadin) to 55 (myosin). Angiotensin-I-converting enzyme (ACE-I) inhibitory peptide sequences were found in abundance in both GEP and dietary proteins. The GEP mucin-5AC and the dietary protein myosin were predicted to release the highest number of ACE-I inhibitory peptides (38 and 49 peptides respectively), and were found to be comparable in their potential to release ACE-I inhibitory peptides. Following SIGIT digestion of eleven representative GEP, nineteen novel GEP-derived peptide sequences were selected by applying quantitative structure-activity relationship rules, and were chemically synthesised. Two novel peptides with the amino acid sequences RPCF and MIM, showing dipeptidyl peptidase IV (DPP-IV) inhibitory activity and five novel antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH)- inhibitory and, or ferric reducing antioxidant power (FRAP) activity) peptides with amino acid sequences CCK, RPCF, CRPK, QQCP and DCR were identified. These results indicate that GEP may contain novel bioactive peptide sequences. The potential release of bioactive peptides, from four GEP (trypsin, lysozyme, mucin, and serum albumin) and a dietary protein (chicken albumin), in the gastrointestinal tract (GIT) was investigated using an in vitro digestion model. The in vitro digests were screened for ACE-I-, renin-, platelet-activating factor acetylhydrolase (PAF-AH)-, and DPP-IV-inhibition, and antioxidant activity. All four in vitro GEP digests showed ACE-I inhibition comparable to that of the positive control captopril. In comparison to the unfractionated digests, the enriched fractions (<3 and <10 kDa) of lysozyme and serum albumin showed greater renin-, PAF-AH-, and DPP-IV-inhibition, and antioxidant potential. Over 190 peptide sequences were identified from these fractions using mass spectrometry. Stomach chyme (SC) and jejunal digesta (JD) were collected from growing pigs that were fed a protein-free diet for a period of 3 days. The peptides extracted from SC and JD samples were characterized by SDS-PAGE, and their ACE-I-, DPPH-, and microsomal lipid peroxidation (MLP)- inhibition, FRAP activity determined. Potential bioactive peptides responsible for bioactivity were identified using mass spectrometry. SDS-PAGE analysis showed that all of the samples contained a heterogeneous mixture of peptides. Porcine JD samples inhibited ACE-I and DPPH, while SC samples inhibited MLP. Characterization studies identified over 180 peptide sequences from the enriched fractions of SC and JD samples that showed the highest activity. Further, a porcine serum albumin peptide sequence (FAKTCVADESAENCDKS) was found to be a sub-sequence of a larger sequence identified in the in vitro digest of human serum albumin. There was considerable inter-animal variation for the bioactivities. This may be attributed to sampling effects and, or natural variations in the gut contents, thus underlining the complexity involved in in vivo release of bioactive peptides. Together, the results indicate: 1) GEP contain abundant encrypted bioactive peptide sequences; 2) GEP-derived bioactive peptides display a range of bioactivities; 3) GEP-derived bioactive peptides are released during gastrointestinal digestion in pigs; 4) GEP may contain numerous novel bioactive peptide sequences encoded within their primary sequence. In conclusion, the evidence reported here suggests that, like the dietary proteins, GEP are also a potentially rich source of exogenously-derived bioactive peptides in the gastrointestinal tract. Beyond their primary functions, GEP may act as an important cryptomic source of bioactive peptides, given that the amount of GEP secreted into the gut is equal to or greater than the dietary protein ingested per day, and that up to 80% of GEP are known to be digested