Peptidomic Approach Research Articles

Peptide profiles and antioxidant capacity of extensive hydrolysates of milk protein concentrate.

Milk protein concentrate was hydrolyzed using one-step enzymatic hydrolysis. Both the peptide profiles and antioxidant activities of the resulting extensive hydrolysates of milk protein concentrate (EMPH) were analyzed using a peptidomics approach based on liquid chromatography-tandem mass spectrometry. The results demonstrated that the degrees of hydrolysis of the 4 EMPH by Alcalase-Protamex, Alcalase-Protease A 2SD, Alcalase-Flavorzyme, and Alcalase-ProteAXH were 12.02%, 16.85%, 15.87%, and 15.77%, respectively. Using size exclusion chromatography, 99.85% of the peptides in the Alcalase-Protease A 2SD hydrolysate were shown to have a molecular weight of <3 kDa. A total of 33 common peptides were identified in the EMPH by liquid chromatography-tandem mass spectrometry, 16 of which were identified as bioactive peptides using bioinformatics. The peptide profiles and the coverage of master proteins of the 4 EMPH were different. The EMPH also exhibited strong free radical scavenging capacity, as indicated by the results of the 1,1-diphenyl-2-picrylhydrazyl radical, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), hydroxyl radical, and reducing power assays. The results of this study provided useful information on the peptide profiles and antioxidant activity of EMPH.

Fermentation of whey protein concentrate by Streptococcus thermophilus strains releases peptides with biological activities

Cheese whey is the principal by-product of cheese making and is perceived as an environmental pollutant. Different technological procedures have been applied to convert whey into value-added products including whey protein concentrate (WPC). In the present study, 2 Lactobacillus helveticus strains and 5 Streptococcus thermophilus strains, isolated from natural whey starter used in Parmigiano Reggiano production, have been evaluated for their capacity to ferment WPC and to release bioactive peptides. WPC fermented with S. thermophilus RBC06 showed the highest antioxidant activity as well as angiotensin-converting enzyme- and dipeptidyl-peptidase-IV-inhibitory activities followed by WPC fermented by S. thermophilus RBC20 and RBN16. An untargeted peptidomics approach was applied to profile the peptides released after WPC fermentation. Several bioactive peptides, mainly angiotensin-converting enzyme inhibitors, were identified and the anti-hypertensive lactotripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) were quantified. S. thermophilus RBC06 produced the highest amount of VPP (12.80 ± 0.45 mg/L) whereas the highest quantity of IPP (0.56 ± 0.02 mg/L) was detected in WPC fermented with S. thermophilus RBN16. The present study paved the way for the further exploitation of S. thermophilus RBC06 as new bioactive-peptide producer and for the application of WPC hydrolysates as additives for designing whey-based beverages with improved healthy properties.

A Peptidomic Approach to Identify Novel Antigen Biomarkers for the Diagnosis of Tuberculosis.

BackgroundHere, we conducted a peptidomic study in murine model to identify novel antigen biomarkers for the diagnosis of tuberculosis (TB) with improved performance.MethodsFour recombinant proteins, including Mycobacterium tuberculosis protein 32 (MPT32), Mycobacterium tuberculosis protein 64 (MPT64), culture filtrate protein 10 (CFP10), and phosphate ABC transporter substrate-binding lipoprotein (PstS1) were expressed and intravenously injected into BALB/c mice. The serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of candidate peptides in serum of suspected TB patients were determined using competitive enzyme-linked immunosorbent assay.ResultsA total of 65 peptides from 4 MTB precursor recombinant proteins were identified in mouse serum by LC-MS/MS, of which 5 peptides were selected as candidates for serological analysis. The concentrations of peptides MPT64-2, CFP10-2 and PstS1-2 in TB patients were significantly higher than those in non-TB patients. MPT64-2 exhibited the most promising sensitivity (81.4%), followed by PstS1-2 and CFP10-2. In addition, PstS1-2 had the highest specificity (93.3%), followed by CFP10-2 and MPT64-2. According to the area under the curve (AUC), MPT64-2 (AUC = 0.863), PstS1-2 (AUC = 0.812) and CFP10-2 (AUC = 0.809) exhibited better diagnostic validity.ConclusionWe develop an effective approach to identify new antigen biomarkers via LC-MS/MS-based peptidomics. Multiple peptides exhibit promising efficacy in diagnosis of active TB patients.

Comprehensive Identification of Short and Medium-Sized Peptides from Pixian Broad Bean Paste Protein Hydrolysates Using UPLC-Q-TOF-MS and UHPLC-Q Exactive HF-X.

Pixian broad bean paste (PBBP) is an indispensable food widely used in many East Asian countries, yet the knowledge about bioactive peptides released from parent proteins by enzymatic hydrolysis is limited. A total of 5867 low-molecular weight peptides were identified in the highly bioactive subfractions of the PBBP alcalase hydrolysates using traditional and peptidomics approaches. 19 short peptides (3-5 amino acids) were identified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry, including 5 tripeptides, 8 tetrapeptides, and 6 pentapeptides. 5848 medium-sized peptides (6-10 amino acids) were characterized using the peptidomics approach, including 1484 hexapeptides, 1217 heptapeptides, 1634 octapeptides, 927 nonapeptides, and 586 decapeptides. The comprehensive method can be used for the investigation of bioactive peptides in complex food matrices.

Peptidomic Approaches and Observations in Neurodegenerative Diseases.

Mass spectrometry (MS), with its immense technological developments over the last two decades, has emerged as an unavoidable technique in analyzing biomolecules such as proteins and peptides. Its multiplexing capability and explorative approach make it a valuable tool for analyzing complex clinical samples concerning biomarker research and investigating pathophysiological mechanisms. Peptides regulate various biological processes, and several of them play a critical role in many disease-related pathological conditions. One important example in neurodegenerative diseases is the accumulation of amyloid-beta peptides (Aβ) in the brain of Alzheimer’s disease (AD) patients. When investigating brain function and brain-related pathologies, such as neurodegenerative diseases, cerebrospinal fluid (CSF) represents the most suitable sample because of its direct contact with the brain. In this review, we evaluate publications applying peptidomics analysis to CSF samples, focusing on neurodegenerative diseases. We describe the methodology of peptidomics analysis and give an overview of the achievements of CSF peptidomics over the years. Finally, publications reporting peptides regulated in AD are discussed.

Mass spectrometry in the discovery of peptides involved in intercellular communication: From targeted to untargeted peptidomics approaches.

Endogenous peptide hormones represent an essential class of biomolecules, which regulate cell-cell communications in diverse physiological processes of organisms. Mass spectrometry (MS) has been developed to be a powerful technology for identifying and quantifying peptides in a highly efficient manner. However, it is difficult to directly identify these peptide hormones due to their diverse characteristics, dynamic regulations, low abundance, and existence in a complicated biological matrix. Here, we summarize and discuss the roles of targeted and untargeted MS in discovering peptide hormones using bioassay-guided purification, bioinformatics screening, or the peptidomics-based approach. Although the peptidomics approach is expected to discover novel peptide hormones unbiasedly, only a limited number of successful cases have been reported. The critical challenges and corresponding measures for peptidomics from the steps of sample preparation, peptide extraction, and separation to the MS data acquisition and analysis are also discussed. We also identify emerging technologies and methods that can be integrated into the discovery platform toward the comprehensive study of endogenous peptide hormones.

Differentiating ultra-high temperature milk and reconstituted milk using an untargeted peptidomic approach with chemometrics

An untargeted peptide profiling based on ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry with chemometrics was performed to differentiate ultra-high temperature processed milk and reconstituted milk. Thirty-three marker peptides were identified, primarily released from the C- or N-terminal of β-casein and αs1-casein. These peptides were produced by heating and protease hydrolysis. Additional heating and storage time experiments showed that the level of 18 marker peptides increased with heat load and storage time, whereas 15 peptides were solely influenced by heat load. The peptides from β-casein showed higher sensitivity to thermal stress compared to those from αs1-casein. Additionally, eight modified peptides of casein were identified as indicators of milk thermal processing. The identified marker peptides can distinguish ultra-high temperature processed milk and reconstituted milk, and are suitable for monitoring heating processes and storage of milk.

In vitro oxidation promoted by sarafloxacin antibiotic residues on myosin and chicken meat proteins

Fluoroquinolones are widely employed to treat or prevent respiratory diseases in poultry, and their residues could be present even after slaughtering. Thus, this study aimed to assess the oxidative effect of fluoroquinolones on myosin and chicken meat proteins under in vitro conditions, using proteomics and peptidomics approaches. Myosin was initially exposed to ciprofloxacin and sarafloxacin, which showed an increase in protein carbonylation over time, especially with sarafloxacin. Then, chicken meat was skipped with sarafloxacin to assess its oxidative effect on in vitro gastrointestinal digestion, and the main proteins were isolated and subsequently identified by mass spectrometry in tandem. The proteins more susceptible to oxidation by sarafloxacin were myosin, β-enolase, CK-M-type, and actin. After peptidomics analysis, collagen peptides resulted as most prone to oxidative damage. These findings confirm the potential negative effects of sarafloxacin residues on chicken meat which could affect its nutritional and technological properties.

Scavenging peptides, antioxidant activity, and hypoglycemic activity of a germinated amaranth (Amaranthus hypochondriacus L.) beverage fermented by Lactiplantibacillus plantarum.

Amaranthus hypochondriacus L. is a pseudocereal with nutritional properties. Some bioactive peptides have been identified from amaranth protein isolates. The metabolic reactions developed during seed germination have produced different functional foods. The present research aimed to develop a non-dairy germinated amaranth-based functional beverage fermented by Lactiplantibacillus plantarum (LP) strain using Lacticaseibacillus casei Shirota (LCS) as control. The content of betalains (BT), total phenolic compounds (TFC), antioxidant capacity (DPPH, ABTS, FRAP), color changes, and scavenging bioactive peptides were determined. BT in the original base was significantly increased after fermentation by LP and LCS (from 1.276±0.030 to 2.732±0.196 and 1.904±0.760mg/100ml, respectively). TFC increased after fermentation; however, no significant differences were found between the two strains (p>.05). The fermentation did not decrease the antioxidant content of the germinated amaranth base. However, a slight increase in the antioxidant capacity was found by DPPH, ABTS, and FRAP in the beverage fermented by LP compared with the LCS one. Moreover, the peptidomic approach suggested 18 peptides with scavenging activity. Thus, a bioactive food product with antioxidant properties was obtained by germination of A. hypochondriacus and its subsequent fermentation by LP. PRACTICAL APPLICATIONS: Non-dairy fermented beverages are novel carriers for probiotics and beneficial metabolites. This research evaluated the antioxidant and hypoglycemic activity of a fermented drink made with amaranth (Amaranthus hypochondriacus) and a potential probiotic strain (Lactiplantibacillus plantarum). The results led to conclude that it is possible to develop functional drinks with potential antioxidant and hypoglycemic activities and provide the biochemical basis for further research and development.

Impact of Tannin Supplementation on Proteolysis during Post-Ruminal Digestion in Wethers Using a Dynamic In Vitro System: A Plant (Medicago sativa) Digestomic Approach.

The aim of this study was to characterize the effects of tannins on plant protein during sheep digestion using a digestomic approach combining in vivo (rumen) conditions and an in vitro digestive system (abomasum and small intestine). Ruminal fluid from wethers infused with a tannin solution or water (control) was introduced into the digester, and protein degradation was followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tannin infusion in the rumen led to a clear decrease in protein degradation-related fermentation end-products, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) protein was more abundant than in control wethers. In the simulated abomasum, peptidomic analysis showed more degradation products of RuBisCo in the presence of tannins. The effect of RuBisCo protection by tannins continued to impact Rubisco digestion into early-stage intestinal digestion but was no longer detectable in late-stage intestinal digestion. The peptidomics approach proved a potent tool for identifying and quantifying the type of protein hydrolyzed throughout the gastrointestinal tract.

Novel ACE inhibitory, antioxidant and α-glucosidase inhibitory peptides identified from fermented rubing cheese through peptidomic and molecular docking

Our previous studies employed the fermented acidified technology to process fermented rubing cheese (FRB) with a self-screened lactic acid bacteria starter. The present study aimed to use the peptidomics approach to identify novel ACEIPs (angiotensin I-converting enzyme inhibitory peptides), AOPs (antioxidant peptides), and α-glucosidase inhibitory peptides from FRB and clarify the molecular interaction mechanism. Water-soluble peptides from FRB had strong ACEI, antioxidant, and α-glucosidase inhibitory activities. LC-MS/MS analyses identified a total of 430 peptides from FRB. Among the identified peptides, six ACEIPs, five AOPs, and five α-glucosidase inhibitory peptides were screened with potential bioactivity via bioinformatics analysis. Their biological activities were evaluated via chemical synthesis and in vitro activity determination. A novel EKVNELSKD demonstrated the strongest ACEI activity (IC50 = 98 μM). Molecular docking suggested that EKVNELSKD formed hydrogen bonds with the S2 (Gln 281 and His513) of ACE. A novel α-glucosidase inhibitor peptide QPHQPLPP (IC50 = 0.821 mg/mL) formed five strong hydrogen bonds with Arg428, Trp710, Asp568, Glu771, and Asn448 in α-glucosidase. Meanwhile, a novel AOP YPFPGPIH (IC50 = 0.402 mg/mL) also showed significant cytoprotective effects against H2O2-induced HepG2 cellular oxidative damage. Furthermore, YPFPGPIH induced antioxidant mechanisms by activating the Keap1-Nrf 2 pathway.

Protein cycling in the eastern tropical North Pacific oxygen‐deficient zone: A de novo‐discovery peptidomic approach

AbstractPeptides are identified using a de novo‐discovery approach in suspended and sinking particles from the eastern tropical North Pacific oxygen‐deficient zone (ODZ) and in a culture of a dominant autotroph from the region, the cyanobacterium Prochlorococcus. The benchmarking experiment with Prochlorococcus shows de novo peptides to be taxonomically specific, and thus of value in augmenting database‐driven approaches. Analysis of the suspended and sinking particles using the de novo‐discovery approach reveals the presence of fungal proteins in deep sinking particles that were not in our original search database, contributing to growing recognition that fungi may play important roles in marine organic matter cycling. Cyanobacterial peptides that have been post‐translationally modified were tracked to depth, where they contribute ~ 1% of the phylum‐level identifiable peptide pool in the sediment trap sample. The majority of peptides found at depth in the detrital pool are associated with membranes, indicating that cellular location is associated with early preservation within the detrital pool. Modified amino acids in sinking and suspended particles include high levels of deamidation, suggesting that partial extracellular degradation of protein could fuel observed anammox and contribute to pools of refractory organic nitrogen.

Structure-function relationship of fermented skate skin gelatin-derived bioactive peptides: a peptidomics approach.

The online version contains supplementary material available at 10.1007/s10068-021-00998-6.

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

SUMMARYWe perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extra-cranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.

A combined metabolomics and peptidomics approach to discriminate anomalous rind inclusion levels in Parmigiano Reggiano PDO grated hard cheese from different ripening stages

Parmigiano Reggiano is a hard cheese with a Protected Designation of Origin (PDO) certification that also applies to the grated product. The percentage of rind in grated Parmigiano Reggiano is regulated by the PDO production Specification and must not exceed the limit of 18% (w/w). The present study evaluates the potential of an untargeted foodomics approach to detect anomalous inclusions of rind in grated Parmigiano Reggiano cheese. In particular, a combined metabolomics and peptidomics approach was used to detect potential markers of counterfeits (rind > 18%). In the framework of realistic food integrity purposes, non-Parmigiano Reggiano grated samples and different ripening times were also considered. Untargeted metabolomics allowed detecting 347 compounds, with a prevalence of amino acids and peptide derivatives, followed by fatty acyls and other compounds (such as lactones, ketones, and aldehydes) typically related to proteolysis and lipolysis events. Overall, the unsupervised multivariate statistics showed that the ripening time plays a hierarchically higher impact than rind inclusion in determining the main differences in the chemical profiles detected. Interestingly, supervised statistics highlighted distinctive markers for ripening time and rind inclusion, with only 16 common discriminant compounds being shared between the two conditions. The best markers of rind inclusion > 18% were 2-hydroxyadenine (VIP score = 1.937; AUC value = 0.83) and the amino acid derivatives argininic acid (VIP score = 1.462; AUC value = 0.75) and 5-hydroxyindole acetaldehyde (VIP score = 1.710; AUC value = 0.86). Interestingly, the medium-chain aldehyde 4-hydroperoxy-2-nonenal was a common marker of both ripening time and anomalous rind inclusion (>18%), likely arising from the lipid oxidation processes. Finally, among potential marker peptides of rind inclusion, the alpha-S1 casein proteolytic product (F)FVAPFPEVFGK(E) could be identified.

Changes of antioxidative activities and peptidomic patterns of Auxenochlorella pyrenoidosa protein hydrolysates: Effects of enzymatic hydrolysis and decoloration processes

Enzymatic hydrolysis and decoloration are indispensable processes for the production of biological peptides from microalgae materials. Herein, this study aimed to investigate the effects of enzymatic hydrolysis and activated carbon decoloration on antioxidative activities and peptidomic patterns of microalgae protein hydrolysates based on a peptidomic approach. Protein hydrolysates with different hydrolysis time (2–16 h) showed significantly different antioxidative activities based on DPPH and ABTS assays. Regarding the peptidomic pattern, principal component analysis (PCA) models revealed that hydrolysates were classified into discriminable clusters depending on the hydrolysis and decoloration processes. Protein hydrolysates contained many aromatic amino acid containing peptides, such as GDSLYPG, SADPETF, FPALN, FPGDY, and LTDWV, contributing to the classification of PCA models. Activated carbon decoloration at the concentration of 5 mg/mL significantly increased the color lightness by 38.20%, reduced antioxidative activities by 29.78% and 33.59% based on DPPH and ABTS methods, and reduced relative abundances of peptides, especially aromatic amino acid containing peptides. Fifteen selected peptides showed strong ABTS radical scavenging abilities and ten of these peptides were correlated with the antioxidative properties of hydrolysates. The monitoring of dynamic production processes at a molecular scale would contribute to the production and quality control of bioactive peptides from Auxenochlorella pyrenoidosa.

Quantitative Peptidomics of Mouse Brain After Infection With Cyst-Forming Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite capable of establishing persistent infection within the host brain and inducing severe neuropathology. Peptides are important native molecules responsible for a wide range of biological functions within the central nervous system. However, peptidome profiling in host brain during T. gondii infection has never been investigated. Using a label-free peptidomics approach (LC–MS/MS), we identified a total of 2,735 endogenous peptides from acutely infected, chronically infected and control brain samples following T. gondii infection. Quantitative analysis revealed 478 and 344 significantly differentially expressed peptides (DEPs) in the acute and chronic infection stages, respectively. Functional analysis of DEPs by Gene Ontology suggested these DEPs mainly originated from cell part and took part in cellular process. We also identified three novel neuropeptides derived from the precursor protein cholecystokinin. These results demonstrated the usefulness of quantitative peptidomics in determining bioactive peptides and elucidating their functions in the regulation of behavior modification during T. gondii infection.

Production and Characterization of Medium-Sized and Short Antioxidant Peptides from Soy Flour-Simulated Gastrointestinal Hydrolysate.

Soybeans (Glycine max) are an excellent source of dietary proteins and peptides with potential biological activities, such as antihypertensive, anti-cholesterol, and antioxidant activity; moreover, they could prevent cancer. Also, soy contains all the essential amino acids for nutrition; therefore, it represents an alternative to animal proteins. The goal of this paper was the comprehensive characterization of medium-sized and short peptides (two to four amino acids) obtained from simulated gastrointestinal digestion. Two different analytical approaches were employed for peptide characterization, namely a common peptidomic analysis for medium-sized peptides and a suspect screening analysis for short peptides, employing an inclusion list of exact m/z values of all possible amino acid combinations. Moreover, fractionation by preparative reversed-phase liquid chromatography was employed to simplify the starting protein hydrolysate. Six fractions were collected and tested for antioxidative activity by an innovative antioxidant assay on human gastric adenocarcinoma AGS cell lines. The two most active fractions (2 and 3) were then characterized by a peptidomic approach and database search, as well as by a suspect screening approach, in order to identify potential antioxidant amino acid sequences. Some of the peptides identified in these two fractions have been already reported in the literature for their antioxidant activity.

Isolation and characterization of cyclotides from the leaves of Viola odorata L. using peptidomic and bioinformatic approach.

The online version contains supplementary material available at 10.1007/s13205-021-02763-2.

Peptidomics of Haemonchus contortus.

The nematode Haemonchus contortus (the barber’s pole worm) is an endoparasite infecting wild and domesticated ruminants worldwide. Widespread anthelmintic resistance of H. contortus requires alternative strategies to control this parasite. Neuropeptide signaling represents a promising target for anthelmintic drugs. Identification and relative quantification of nematode neuropeptides are, therefore, required for the development of such therapeutic targets. In this work, we undertook the profiling of the whole H. contortus larvae at different stages for the direct sequencing of the neuropeptides expressed at low levels in these tissues. We set out a peptide extraction protocol and a peptidomic workflow to biochemically characterize bioactive peptides from both first-stage (L1) and third-stage larvae (L3) of H. contortus. This work led to the identification and quantification at the peptidomic level of more than 180 mature neuropeptides, including amidated and nonamidated peptides, arising from 55 precursors of H. contortus. The differential peptidomic approach provided evidence that both life stages express most FMRFamide-like peptides (FLPs) and neuropeptide-like proteins (NLPs). The H. contortus peptidome resource, established in this work, could add the discovery of neuropeptide system-targeting drugs for ruminants.