Peptidomic Approach Research Articles

Peptidomic strategy for purification and identification of potential ACE-inhibitory and antioxidant peptides in Tetradesmus obliquus microalgae.

Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity. Graphical abstract Workflow showing the entire peptidomic approach developed for identification of bioactive peptides in microalgae.

Identification of Endogenous Neuropeptides in the Nematode C. elegans Using Mass Spectrometry.

The nematode Caenorhabditis elegans lends itself as an excellent model organism for peptidomics studies. Its ease of cultivation and quick generation time make it suitable for high-throughput studies. Adult hermaphrodites contain 959 somatic nuclei that are ordered in defined, differentiated tissues. The nervous system, with its 302 neurons, is probably the most known and studied endocrine tissue. Moreover, its neuropeptidergic signaling pathways display a large number of similarities with those observed in other metazoans. However, various other tissues have also been shown to express several neuropeptides. This includes the hypodermis, gonad, gut, and even muscle. Hence, whole mount peptidomics of C. elegans cultures provides an integral overview of peptidergic signaling between the different tissues of the entire organism. Here, we describe a peptidomics approach used for the identification of endogenous (neuro)peptides in C. elegans. Starting from a detailed peptide extraction procedure, we will outline the setup for an online liquid chromatography-mass spectrometry (LC-MS) analysis and describe subsequent data analysis approaches.

Affinity Purification of Neuropeptide Precursors from Mice Lacking Carboxypeptidase E Activity.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide-processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. Not all peptides with basic C-terminal residues within a cell are CPE substrates, and these other peptides will also be purified on the anhydrotrypsin affinity column. However, a comparison of peptides purified from wild-type mice and from mice lacking CPE allows for the rapid identification of CPE substrates based on their large increase in the absence of CPE.

Peptidomic Identification of Cysteine-Rich Peptides from Plants.

Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.

Non-targeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-Amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a non-targeted manner.

Development of an enrichment method for endogenous phosphopeptide characterization in human serum.

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods. Thus, in this paper, different phosphopeptide enrichment strategies were pursued, based either on metal oxide affinity chromatography (MOAC, in the form of commercial TiO2 spin columns or magnetic graphitized carbon black-TiO2 composite), or on immobilized metal ion affinity chromatography (IMAC, in the form of Ti4+-IMAC magnetic material or commercial Fe3+-IMAC spin columns). While MOAC strategies proved completely unsuccessful, probably due to interfering phospholipids displacing phosphopeptides, the IMAC materials performed very well. Different sample preparation strategies were tested, comprising direct dilution with the loading buffer, organic solvent precipitation, and lipid removal from the matrix, as well as the addition of phosphatase inhibitors during sample handling for maximized endogenous phosphopeptide enrichment. All data were acquired by a shotgun peptidomics approach, in which peptide samples were separated by reversed-phase nanoHPLC hyphenated with high-resolution tandem mass spectrometry. The devised method allowed the identification of 176 endogenous phosphopeptides in fresh serum added with inhibitors by the direct dilution protocol and the Ti4+-IMAC magnetic material enrichment, but good results could also be obtained from the commercial Fe3+-IMAC spin column adapted to the batch enrichment protocol.

Structure and function of a novel antioxidant peptide from the skin of tropical frogs.

The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin-I, has a molecular weight [M+H]+ = 1543.69Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin-I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin-I has a novel primary sequence with low similarity compared with previously described amphibian's AOPs. Antioxidin-I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin-I presented a low cytotoxicity and suppressed menadione-induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia-induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide.

Identification of a moronecidin-like antimicrobial peptide in the venomous fish Pterois volitans: Functional and structural study of pteroicidin-α

The present study characterizes for the first time an antimicrobial peptide in lionfish (Pterois volitans), a venomous fish. Using a peptidomic approach, we identified a mature piscidin in lionfish and called it pteroicidin-α. We detected an amidated form (pteroicidin-α- CONH2) and a non-amidated form (pteroicidin-α-COOH), and then performed their functional and structural study. Interestingly, the two peptides displayed different antibacterial and hemolytic activity levels. Pteroicidin-α-CONH2 was bactericidal on human pathogens like Staphylococcus aureus or Escherichia coli, as well as on the fish pathogen Aeromonas salmonicida, while pteroicidin-α-COOH only inhibited their growth. Furthermore, the two peptides induced hemolysis of red blood cells from different vertebrates, namely humans, sea bass and lesser-spotted dogfish. Hemolysis occurred with low concentrations of pteroicidin-α-CONH2, indicating greater toxicity of the amidated form. Circular dichroism analysis showed that both peptides adopted a helical conformation, yet with a greater α-helix content in pteroicidin-α-CONH2. Overall, these results suggest that amidation strongly influences pteroicidin-α by modifying its structure and its physico-chemical characteristics and by increasing its hemolytic activity.

Peptidomic as a tool for assessing protein digestion

One way to strengthen our knowledge on the effect of food on human health is to better understand the mechanisms of food disintegration in the gastrointestinal tract. During digestion, dietary proteins are hydrolyzed by digestive enzymes in the stomach and small intestine into a myriad of peptides. Peptidomic approaches have been developed in order to identify the peptides released in the lumen during in vitro and in vivo digestion by mainly using mass spectrometry. It has therefore been possible to demonstrate that peptides known to exert biological activities are present in the lumen at concentrations compatible with a biological action. However, this does not prove that the detected bioactive peptides have an effect on human health and more work is needed to determine peptide bioavailability and lifetime in the bloodstream. Thousands of peptides can be identified unambiguously and some efforts need to be done to develop new strategies of data treatment.

Quantitative measurement of a candidate serum biomarker peptide derived from α2-HS-glycoprotein, and a preliminary trial of multidimensional peptide analysis in females with pregnancy-induced hypertension.

Purpose We previously attempted to develop quantitative enzyme-linked immunosorbent assay (ELISA) systems for the PDA039/044/071 peptides, potential serum disease biomarkers (DBMs) of pregnancy-induced hypertension (PIH), primarily identified by a peptidomic approach (BLOTCHIP®-mass spectrometry (MS)). However, our methodology did not extend to PDA071 (cysteinyl α2-HS-glycoprotein341-367), due to difficulty to produce a specific antibody against the peptide. The aim of the present study was to establish an alternative PDA071 quantitation system using liquid chromatography-multiple reaction monitoring (LC-MRM)/MS, to explore the potential utility of PDA071 as a DBM for PIH. Methods We tested heat/acid denaturation methods in efforts to purify serum PDA071 and developed an LC-MRM/MS method allowing for specific quantitation thereof. We measured serum PDA071 concentrations, and these results were validated including by three-dimensional (3D) plotting against PDA039 (kininogen-1439-456)/044 (kininogen-1438-456) concentrations, followed by discriminant analysis. Results PDA071 was successfully extracted from serum using a heat denaturation method. Optimum conditions for quantitation via LC-MRM/MS were developed; the assayed serum PDA071 correlated well with the BLOTCHIP® assay values. Although the PDA071 alone did not significantly differ between patients and controls, 3D plotting of PDA039/044/071 peptide concentrations and construction of a Jackknife classification matrix were satisfactory in terms of PIH diagnostic precision. Conclusions Combination analysis using both PDA071 and PDA039/044 concentrations allowed PIH diagnostic accuracy to be attained, and our method will be valuable in future pathophysiological studies of hypertensive disorders of pregnancy.

Evolution of oxidised peptides during the processing of 9 months Spanish dry-cured ham

Sensory and nutritional properties of dry-cured ham can be negatively affected due to oxidative modifications of muscle proteins during its processing. In this study, a peptidomic approach has been used in order to study the evolution of oxidised peptides generated throughout the dry-curing process (0, 2, 3.5, 5, 6.5 and 9months), focusing on those derived from major myofibrillar proteins. A total of 67 peptides showing methionine, proline, and tryptophan oxidations were identified in common in all samples by nano liquid chromatography coupled to tandem mass spectrometry for their relative quantification using a label-free methodology, showing the hydrolysis of some of the peptides during the process. So, the peptidomics strategy used in this work has resulted to be very useful as a complementary tool to the methods currently used to study protein oxidation, allowing a better understanding of the oxidation at peptide level and the influence of ham processing conditions.

KL 10 Animal models for gestational hypertension

The prevalence of cardiovascular diseases and particularly of hypertension is increasing worldwide. Consequently, the number of women with hypertension in pregnancy is also increasing. Several rodent models of hypertension have been characterised in depth but only few models exhibit the complex cardiovascular phenotype that is typically seen in human hypertension. The stroke-prone spontaneously hypertensive rat (SHRSP) is characterised by hypertension, organ damage including left ventricular hypertrophy and stroke, metabolic disorders and a more severe phenotype in male compared to female rats. Whilst the majority of research in rodent pregnancy focusses on models of pre-eclampsia we are particularly interested in the adaptation to pregnancy in pre-existing hypertension. As such, the SHRSP is an ideal model for our studies. We have recently described important features of pregnant SHRSP that will help to understand human hypertensive pregnancy. We have demonstrated impaired uterine artery remodelling and reduced uteroplacental blood flow compared to the normotensive Wistar Kyoto strain (WKY), paralleled by impaired uterine artery endothelial function. Vascular changes in the pregnant SHRSP are associated with a unique inflammatory profile resulting in increased placental and systemic TNF-alpha levels. The TNF-alpha receptor antagonist etanercept improves uterine artery function and reduces blood pressure in the pregnant SHRSP. Natural Killer (NK) cells appear to contribute to placental TNF-alpha production in the SHRSP and etanercept causes a change in cytotoxicity of these NK cells. We are currently exploring how an even more severe hypertensive phenotype in the SHRSP affects pregnancy outcome and if we can observe features of superimposed pre-eclampsia in this model. To this end, we expose pregnant SHRSP to high salt diet or angiotensin II. In parallel to hypothesis driven experiments we perform unbiased screens in the pregnant and non-pregnant SHRSP, focussing on transcriptomic, peptidomic and proteomic approaches. In summary, the SHRSP is an excellent model to study the effects of chronic hypertension on the physiological changes associated with pregnancy.

A cold-induced phytosulfokine peptide is related to the improvement of loquat fruit chilling tolerance

A novel peptidomics approach was used to discover cold-induced peptides in loquat fruit. Twenty unique peptides derived from 18 proproteins were identified, and they were involved in sugar signalling, protein metabolism and stress response. The quantitative analysis revealed 7 peptides with more than 2-fold upregulation, especially a 4.96-fold increase detected in the phytosulfokine (PSK) peptide. To further evaluate effects of PSK1 on fruit chilling tolerance, weight loss, firmness and internal browning were investigated in PSK1-treated loquat fruit at 0°C. By contrast, these chilling injury symptoms were effectively reduced by PSK1. PSK1 markedly delayed decreases of ATP content and energy charge. The PSK1-treated fruit exhibited significantly lower activities of cell-wall degrading enzymes and transcripts of genes related to lignin synthesis. Our results demonstrated that PSK1 improves chilling tolerance of loquat fruit by maintaining high energy status and cell integrity. Peptidomics analysis provides a promising tool to discover some key peptides.

Peptidomics as a tool for characterizing bioactive milk peptides.

Food peptidomics is a sub-field of proteomics that focuses on the composition, interactions, and properties of bioactive peptides present in different food matrices. The milk peptidome is considered a valuable source of a number of biologically active peptides. Increasing use of peptidomic techniques-including the application of high-resolution techniques, such as mass spectrometry-has led to enhancements of our knowledge regarding the health benefits of dairy products, as well as improved monitoring for food control and food safety. Chromatographic techniques, both at the analytical and preparative scale, are used also in the identification of novel peptides, including those synthesized and those obtained through fermentation processes. The present review focuses on peptidomic approaches to the investigation of bioactive milk peptides, including bioinformatics, chemometric tools, and proteomic/peptidomic methods.

Novel Kazal-type proteinase inhibitors from the skin secretion of the Splendid leaf frog, Cruziohyla calcarifer

Peptidase inhibitors have an important role controlling a variety of biological processes. Here, we employed a peptidomic approach including molecular cloning, tandem mass spectrometry and enzymatic assays to reveal 7 Kazal-type proteinase inhibitors (CCKPs) (18 variants) in the skin secretion of the unexplored frog, Cruziohyla calcarifer. All 18 proteins shared the Kazal pattern C-X(7)-C-X(6,7)-C-X(6,7)-Y-X(3)-C-X(2)-C-X(15-21)-C and 3 disulphide bridges. Based on structural comparative analysis, we deemed trypsin and chymotrypsin inhibitory activity in CCKP-1, 4 and CCKP 2, 5, 7, respectively. These peptidase inhibitors presumably play a role to control the balance between other functional peptides produced in the amphibian skin secretions.

Discovery of lung squamous carcinoma biomarkers by profiling the plasma peptide with LC/MS/MS

Biomarkers can be used for the screening and clinical diagnosis of cancer, and peptidomics approach has been proven successful in the research of biomarkers. To develop better peptidomic technologies for fast, accurate, and reliable detection of peptides biomarkers for lung cancer, we have improved the procedures of blood collection to minimize the degradation of the blood proteins and optimize the extraction of peptidome peptides from plasma samples based on acetonitrile precipitation associated with size exclusion chromatography (SEC). Studies show that squamous cell carcinomas are found to express CAGE1, SPAT9 and TEX28 genes at significantly higher rates, and the results suggest that as tumors progress, the level of CAGE1, SPAT9 and TEX28 genes are likely to increase and lead to immunization. This suggests a potentially important therapeutic method for cancer testis-based cancer vaccines.

Peptidomic Approach to Developing ELISAs for the Determination of Bovine and Porcine Processed Animal Proteins in Feed for Farmed Animals.

The European Commission (EC) wants to reintroduce nonruminant processed animal proteins (PAPs) safely into the feed chain. This would involve replacing the current ban in feed with a species-to-species ban which, in the case of nonruminants, would only prohibit feeding them with proteins from the same species. To enforce such a provision, there is an urgent need for species-specific methods for detecting PAPs from several species in animal feed and in PAPs from other species. Currently, optical microscopy and the polymerase chain reaction are the officially accepted methods, but they have limitations, and alternative methods are needed. We have developed immunoassays using antibodies raised against targets which are not influenced by high temperature and pressure. These targets were identified in a previous study based on an experimental approach. One optimized competitive ELISA detects bovine PAPs at 2% in plant-derived feed. The detection capability demonstrated on blind samples shows a good correlation with mass spectrometry results.

Follicular fluid biomarkers for human in vitro fertilization outcome: Proof of principle.

BackgroundHuman follicular fluid (FF) is a unique biological fluid in which the oocyte develops in vivo, and presents an optimal source for non-invasive biochemical predictors. Oocyte quality directly influences the embryo development and hence, may be used as a predictor of embryo quality. Peptide profiling of FF and its potential use as a biomarker for oocyte quality has never been reported.MethodsThis study screened FF for peptide biomarkers that predict the outcome of in vitro fertilization (IVF). Potential biomarkers were discovered by investigating 2 training datasets, consisting both of 17 samples and validating on an independent experiment containing 32 samples. Peptide profiles were acquired by nano-scale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS).ResultsFrom the training datasets 53 peptides were found as potential biomarker candidates, predicting the fertilization outcome of 24 out of the 32 validation samples blindly (81.3% sensitivity, 68.8% specificity, AUC = 0.86). Seven potential biomarker peptides were identified. They were derived from: insulin-like growth factor binding protein-5, alpha-2-antiplasmin, complement component 3, inter-alpha-trypsin inhibitor heavy chain H1, serum albumin, protein diaphanous homolog 1 and plastin-3.ConclusionsThe MS-based comprehensive peptidomic approach carried out in this study, established a novel panel of potential biomarkers that present a promising predictive accuracy rate in fertilization outcome, and indicates FF as an interesting biomarker resource to improve IVF clinic routine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0106-9) contains supplementary material, which is available to authorized users.

Peptidomics of the zebrafish Danio rerio: In search for neuropeptides

(Neuro)peptides are small messenger molecules that are derived from larger, inactive precursor proteins by the highly controlled action of processing enzymes. These biologically active peptides can be found in all metazoan species where they orchestrate a wide variety of physiological processes. Obviously, detailed knowledge on the actual peptide sequences, including the potential existence of truncated versions or presence of post-translation modifications, is of high importance when studying their function. A peptidomics approach therefore aims to identify and characterize the endogenously present peptide complement of a defined tissue or organism using liquid chromatography and mass spectrometry. While the zebrafish Danio rerio is considered as an important aquatic model for medical research, neuroscience, development and ecotoxicology, very little is known about their peptidergic signaling cascades. We therefore set out to biochemically characterize endogenously present (neuro)peptides from the zebrafish brain. This peptidomics setup yielded >60 different peptides in addition to various truncated versions. SignificanceThough the zebrafish is a well-established model organism to study vertebrate biology and gene functions in either a medical or (eco)toxicological context, very little knowledge about neuropeptidergic signaling cascades is available. We therefore set out to characterize endogenously present peptides from the zebrafish brain using a peptidomics setup yielding a total number of 105 peptide identifications. To our knowledge, it is the first attempt to biochemically isolate and characterize neuropeptides from a fish species in a high-throughput manner. This archive of identified endogenous peptides is likely to aid further functional elucidation of defined neuropeptidergic signaling systems (e.g. characterization of cognate G-protein coupled receptors). Furthermore, our methodology allows studying the changes in peptide expression in response to changes in the organism or the environment using differential peptidomics.

A peptidomic approach for the identification of antioxidant and ACE-inhibitory peptides in sardinelle protein hydrolysates fermented by Bacillus subtilis A26 and Bacillus amyloliquefaciens An6

Antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities of sardinelle (Sardinella aurita) protein hydrolysates (SPHs) obtained by fermentation with Bacillus subtilis A26 (SPH-A26) and Bacillus amyloliquefaciens An6 (SPH-An6) were investigated. Both hydrolysates showed dose-dependent antioxidant activities evaluated using various in vitro antioxidant assays. Further, they were found to exhibit ACE-inhibitory activity. Peptides from SPH-A26 and SPH-An6 were analyzed by nESI-LC–MS/MS and approximately 800 peptides were identified. Identified peptides derived mainly from myosin (43% and 31% in SPH-An6 and SPH-A26, respectively). Several peptides identified in both hydrolysates were found to share sequences with previously identified antioxidant and ACE-inhibitory peptides based on Biopep database. Some of these peptides were selected for synthesis and their biological activities were evaluated. Among the synthesized peptides, NVPVYEGY and ITALAPSTM were found to be the most effective ACE-inhibitors with IC50 values of 0.21 and 0.23mM, respectively. On the other hand, NVPVYEGY, which exhibited the highest ACE-inhibitory activity, showed the highest reducing power and peroxyl radical scavenging activities, followed by SLEAQAEKY and GTEDELDKY. The results of this study suggest that fermented sardinelle protein hydrolysates are a good source of natural antioxidant peptides and could have the potential to act as hypotensive nutraceutical ingredients.