Identification Of Peptides Research Articles

DIA-based serum proteomics revealed the protective effect of modified siwu decoction against hypobaric hypoxia

Ethnopharmacological relevanceSiwu decoction (SWD) is a common traditional formula for nourishing blood, and its derived formulas are also widely used in traditional Chinese medicine (TCM) clinic. However, the protective effects of SWD and its derived formulas on blood deficiency and blood stasis caused by rushing to the plateau are rarely reported, and the underlying mechanism has not been fully elucidated. Aim of the studyThis study explores the pharmacological effects and mechanisms of modified siwu decoction (MSWD) adding Persicae Semans (Prunus persica (L.) Batsch) and Carthami Flos (Carthamus tinctorius L.) against hypobaric hypoxia (HH). The acute toxicity of MSWD was also evaluated to further validate the potential of MSWD as a therapeutic candidate for HH. Materials and methodsHypoxic models of C57BL/6 J and KM male mice were used to evaluate the pharmacological effect of MSWD. 2 μL serum sample of C57BL/6 J mice was digested into peptide mixtures and analyzed with DIA mode on an Orbitrap Fusion Lumos mass spectrometer after LC separation. The peptide and protein identifications were limited to a 1% FDR. Screening of differential expressed proteins, correlation analysis, hierarchical clustering analysis, principal components analysis and Mfuzz analysis were all performed by R packages. The protein−protein interaction network was analyzed using the STRING website and constructed with Cytoscape software. ResultsMSWD showed a protective effect against acute hypoxia exposure through increasing the number of red blood cells and improving hemodynamics indexes in mice. Meanwhile, the biochemical results showed that MSWD could reduce the inflammation and oxidative stress, reduce the content of organ injury biomarkers and significantly improve the high-intensity exercise ability of mice. Subsequently, serum DIA proteomic results revealed significant changes in proteomic characteristics after MSWD intervention. Specifically, proteins related to oxidative stress and ubiquitin-proteasome system, such as Sod1, Gstp1, Vcp and Usp14, were down-regulated after MSWD intervention, suggesting that the protective effect of MSWD involved the reduction of oxidative stress and energy expenditure. MSWD also intervened in energy metabolism and lipid metabolism processes by altering the expression levels of Eno1, Sphk1 and Apoa1 to ameliorate hypoxia-induced disorders. At the same time, MSWD acute toxicity test showed no obvious toxicity. ConclusionsMSWD has a good protective effect against HH by ameliorating hypoxia-induced disorders of energy and lipid metabolism, supporting MSWD as a safe drug candidate for the prevention and treatment of acute hypoxia fatigue.

Sage: An Open-Source Tool for Fast Proteomics Searching and Quantification at Scale.

The growing complexity and volume of proteomics data necessitate the development of efficient software tools for peptide identification and quantification from mass spectra. Given their central role in proteomics, it is imperative that these tools are auditable and extensible─requirements that are best fulfilled by open-source and permissively licensed software. This work presents Sage, a high-performance, open-source, and freely available proteomics pipeline. Scalable and cloud-ready, Sage matches the performance of state-of-the-art software tools while running an order of magnitude faster.

Identification of a functional peptide of a probiotic bacterium-derived protein for the sustained effect on preventing colitis

ABSTRACT Several probiotic-derived factors have been identified as effectors of probiotics for exerting beneficial effects on the host. However, there is a paucity of studies to elucidate mechanisms of their functions. p40, a secretory protein, is originally isolated from a probiotic bacterium, Lactobacillus rhamnosus GG. Thus, this study aimed to apply structure-functional analysis to define the functional peptide of p40 that modulates the epigenetic program in intestinal epithelial cells for sustained prevention of colitis. In silico analysis revealed that p40 is composed of a signal peptide (1–28 residues) followed by a coiled-coil domain with uncharacterized function on the N-terminus, a linker region, and a β-sheet domain with high homology to CHAP on the C-terminus. Based on the p40 three-dimensional structure model, two recombinant p40 peptides were generated, p40N120 (28–120 residues) and p40N180 (28–180 residues) that contain first two and first three coiled coils, respectively. Compared to full-length p40 (p40F) and p40N180, p40N120 showed similar or higher effects on up-regulating expression of Setd1b (encoding a methyltransferase), promoting mono- and trimethylation of histone 3 on lysine 4 (H3K4me1/3), and enhancing Tgfb gene expression and protein production that leads to SMAD2 phosphorylation in human colonoids and a mouse colonic epithelial cell line. Furthermore, supplementation with p40F and p40N120 in early life increased H3K4me1, Tgfb expression and differentiation of regulatory T cells (Tregs) in the colon, and mitigated disruption of epithelial barrier and inflammation induced by DSS in adult mice. This study reveals the structural feature of p40 and identifies a functional peptide of p40 that could maintain intestinal homeostasis.

Inclusion of Porous Graphitic Carbon Chromatography Yields Greater Protein Identification and Compartment and Process Coverage and Enables More Reflective Protein-Level Label-Free Quantitation.

The ubiquity of mass spectrometry-based bottom-up proteomic analyses as a component of biological investigation mandates the validation of methodologies that increase acquisition efficiency, improve sample coverage, and enhance profiling depth. Chromatographic separation is often ignored as an area of potential improvement, with most analyses relying on traditional reversed-phase liquid chromatography (RPLC); this consistent reliance on a single chromatographic paradigm fundamentally limits our view of the observable proteome. Herein, we build upon early reports and validate porous graphitic carbon chromatography (PGC) as a facile means to substantially enhance proteomic coverage without changes to sample preparation, instrument configuration, or acquisition methods. Analysis of offline fractionated cell line digests using both separations revealed an increase in peptide and protein identifications by 43% and 24%, respectively. Increased identifications provided more comprehensive coverage of cellular components and biological processes independent of protein abundance, highlighting the substantial quantity of proteomic information that may go undetected in standard analyses. We further utilize these data to reveal that label-free quantitative analyses using RPLC separations alone may not be reflective of actual protein constituency. Together, these data highlight the value and comprehension offered through PGC-MS proteomic analyses. RAW proteomic data have been uploaded to the MassIVE repository with the primary accession code MSV000091495.

Baldur: Bayesian Hierarchical Modeling for Label-Free Proteomics with Gamma Regressing Mean-Variance Trends

Label-free proteomics is a fast-growing methodology to infer abundances in mass spectrometry proteomics. Extensive research has focused on spectral quantification and peptide identification. However, research toward modeling and understanding quantitative proteomics data is scarce. Here we propose a Bayesian hierarchical decision model (Baldur) to test for differences in means between conditions for proteins, peptides, and post-translational modifications. We developed a Bayesian regression model to characterize local mean-variance trends in data, to estimate measurement uncertainty and hyperparameters for the decision model. A key contribution is the development of a new gamma regression model that describes the mean-variance dependency as a mixture of a common and a latent trend—allowing for localized trend estimates. We then evaluate the performance of Baldur, limma-trend, and t test on six benchmark datasets: five total proteomics and one post-translational modification dataset. We find that Baldur drastically improves the decision in noisier post-translational modification data over limma-trend and t test. In addition, we see significant improvements using Baldur over the other methods in the total proteomics datasets. Finally, we analyzed Baldur’s performance when increasing the number of replicates and found that the method always increases precision with sample size, while showing robust control of the false positive rate. We conclude that our model vastly improves over popular data analysis methods (limma-trend and t test) in several spike-in datasets by achieving a high true positive detection rate, while greatly reducing the false-positive rate.

Immunoinformatic Approach for Rational Identification of Immunogenic Peptides Against Host Entry and/or Exit Mpox Proteins and Potential Multiepitope Vaccine Construction.

COVID-19 has intensified humanity's concern about the emergence of new pandemics. Since 2018, epidemic outbreaks of the mpox virus have become worrisome. In June 2022, the World Health Organization declared the disease a global health emergency, with 14 500 cases reported by the Centers for Disease Control and Prevention in 60 countries. Therefore, the development of a vaccine based on the current virus genome is paramount in combating new cases. In view of this, we hypothesized the obtainment of rational immunogenic peptides predicted from proteins responsible for entry of the mpox virus into the host (A17L, A26L/A30L, A33R, H2R, L1R), exit (A27L, A35R, A36R, C19L), and both (B5R). To achieve this, we aligned the genome sequencing data of mpox virus isolated from an infected individual in the United States in June 2022 (ON674051.1) with the reference genome dated 2001 (NC_003310.1) for conservation analysis. The Immune Epitope Database server was used for the identification and characterization of the epitopes of each protein related to major histocompatibility complex I or II interaction and recognition by B-cell receptors, resulting in 138 epitopes for A17L, 233 for A28L, 48 for A33R, 77 for H2R, 77 for L1R, 270 for A27L, 72 for A35R, A36R, 148 for C19L, and 276 for B5R. These epitopes were tested in silico for antigenicity, physicochemical properties, and allergenicity, resulting in 51, 40, 10, 34, 38, 57, 25, 7, 47, and 53 epitopes, respectively. Additionally, to select an epitope with the highest promiscuity of binding to major histocompatibility complexes and B-cell receptor simultaneously, all epitopes of each protein were aligned, and the most repetitive and antigenic regions were identified. By classifying the results, we obtained 23 epitopes from the entry proteins, 16 from the exit proteins, and 7 from both. Subsequently, 1 epitope from each protein was selected, and all 3 were fused to construct a chimeric protein that has potential as a multiepitope vaccine. The constructed vaccine was then analyzed for its physicochemical, antigenic, and allergenic properties. Protein modeling, molecular dynamics, and molecular docking were performed on Toll-like receptors 2, 4, and 8, followed by in silico immune simulation of the vaccine. Finally, the results indicate an effective, stable, and safe vaccine that can be further tested, especially in vitro and in vivo, to validate the findings demonstrated in silico.

Spectroscape enables real-time query and visualization of a spectral archive in proteomics

In proteomics, spectral archives organize the enormous amounts of publicly available peptide tandem mass spectra by similarity, offering opportunities for error correction and novel discoveries. Here we adapt an indexing algorithm developed by Facebook for organizing online multimedia resources to tandem mass spectra and achieve practically instantaneous retrieval and clustering of approximate nearest neighbors in a large spectral archive. An interactive web-based graphical user interface enables the user to view a query spectrum in its clustered neighborhood, which facilitates contextual validation of peptide identifications and exploration of the dark proteome.

Advances in proteomics: characterization of the innate immune system after birth and during inflammation.

Proteomics is the characterization of the protein composition, the proteome, of a biological sample. It involves the large-scale identification and quantification of proteins, peptides, and post-translational modifications. This review focuses on recent developments in mass spectrometry-based proteomics and provides an overview of available methods for sample preparation to study the innate immune system. Recent advancements in the proteomics workflows, including sample preparation, have significantly improved the sensitivity and proteome coverage of biological samples including the technically difficult blood plasma. Proteomics is often applied in immunology and has been used to characterize the levels of innate immune system components after perturbations such as birth or during chronic inflammatory diseases like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). In cancers, the tumor microenvironment may generate chronic inflammation and release cytokines to the circulation. In these situations, the innate immune system undergoes profound and long-lasting changes, the large-scale characterization of which may increase our biological understanding and help identify components with translational potential for guiding diagnosis and treatment decisions. With the ongoing technical development, proteomics will likely continue to provide increasing insights into complex biological processes and their implications for health and disease. Integrating proteomics with other omics data and utilizing multi-omics approaches have been demonstrated to give additional valuable insights into biological systems.

An on-line stop-flow RPLC × SEC-MS/DPPH radical scavenging activity analysis system and its application in separation and identification of antioxidant peptides

Food-derived antioxidant peptides have become the focus of research due to their high safety and low cost. However, the discovery is suffering from a low efficient and empirical approach, involving multi-step off-line separation and identification. In this work, an on-line stop-flow RPLC × SEC-MS/DPPH radical scavenging activity analysis system was developed. For optimization, the conditions: 10 m reaction loop, 200 μM DPPH radical concentration, 40℃ temperature and 0.06 % formic acid were recommended. The system was fully validated by its application in glutathione analysis. The system was further applied in analysis of complex mixed standards, and the dipeptides GC (Gly-Cys) and CW (Cys-Trp) with relatively strong DPPH radical scavenging activity were validated. Maize protein hydrolysates were used for tests and the peptide AC (Ala-Cys) of high probability with strong DPPH radical scavenging activity was identified, demonstrating a high potential of the system. This would help to facilitate the discovery of antioxidative peptides in the future.

Research Progress on the Preparation and Function of Antioxidant Peptides from Walnuts.

Food-derived peptides have good antioxidant activity and are highly safe for humans; consequently, there has been continuous growth in research on antioxidants, with potential applications in food, medicine, cosmetics, and other fields. Among food-derived peptides, walnut-derived peptides have attracted increasing attention as food-derived peptides rich in eight essential amino acids. This review summarizes the progress made in the development and identification of antioxidant peptides in walnut proteins. This article mainly describes the interaction between reactive oxygen species and cellular antioxidant products, modulation of enzyme content and activity, and regulation of the redox signaling pathways and analyzes the mechanisms of reduction in oxidative stress. Finally, the complex structure-activity relationships of walnut-derived peptides are analyzed based on their amino acid composition and secondary structure of the polypeptides. This review provides a theoretical basis for the production of walnut-derived antioxidant peptides and could help promote the development of the walnut industry.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry.

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom. These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

Preparation and identification of a novel peptide with high antioxidant activity from corn gluten meal.

The antioxidant activity of corn peptides is related to their molecular weight and structure. Corn gluten meal (CGM) was hydrolyzed using a combination of Alcalase, Flavorzyme and Protamex, and the hydrolysates were subjected to antioxidant activity analysis after further fractionation. Corn peptides with molecular weights less than 1kDa (CPP1) exhibited excellent antioxidant activity. A novel peptide, Arg-Tyr-Leu-Leu (RYLL), was identified from CPP1. RYLL displayed preferable scavenging capacities for ABTS radicals and DPPH radicals, with IC50 values of 0.122mg/ml and 0.180mg/ml, respectively. Based on quantum calculations, RYLL had multiple antioxidant active sites, and tyrosine was the main active site due to the highest energy of the highest occupied molecular orbit (HOMO). Moreover, the simple peptide structure and hydrogen bond network of RYLL contributed to the exposure of the active site. This study elucidated the antioxidant mechanism of corn peptides, which could provide an understanding for CGM hydrolysates as natural antioxidants.

Identification and characterisation of taste-enhancing peptides from oysters (Crassostrea gigas) via the Maillard reaction.

Oysters, which are flavourful edible marine products, have been utilised to produce Maillard reaction products (MRPs), which contribute to saltiness enhancement. Here, the molecular weight distribution, free amino acids, and taste characteristics of MRPs were analysed, while ultraviolet light was used to observe the Maillard reaction. Both thermal degradation and cross-linking reactions occur during the Maillard reaction. When the Maillard reaction time was 90min, the saltiness, umami, and richness of the MRPs peaked, however bitterness reached its lowest value. Moreover, at an MRP concentration of 1.5mg/mL, salts were reduced by 35.71% in a 3mg/mL sodium chloride solution without reducing saltiness, based on sensory evaluation. Glycation sites of the MRPs, which are crucial for saltiness enhancement and derived from a variety of protein sources, were determined using nano-HPLC-MS/MS analysis. Our study establishes the foundation for preparing salt-enhancing peptides, accelerating the popularisation of oyster-derived flavouring agents.

Identification and action mechanism of novel antioxidative peptides from copra meal protein

This study aimed to identify novel antioxidant peptides from copra meal protein. The protein was enzymatically digested and properties of released peptides were predicted using in silico approach. The antioxidant activity of peptides was determined using ABTS and DPPH assays. Molecular docking and the isothermal titration calorimetry (ITC) were also used to explore the mechanism of antioxidant peptides scavenging free radicals. The results showed that peptides GMEEER and EEGER scavenged ABTS radical cation with IC50 values of 0.46 ± 0.067 mmol/L and 6.54 ± 0.14 mmol/L, and DPPH radicals with IC50 values of 6.98 ± 0.29 mmol/L and 11.06 ± 0.18 mmol/L. Hydrophobic interactions and hydrogen bonding were verified as the major driving force for scavenging of free radicals of peptides GMEEER and EEGER by molecular docking and ITC. Especially, the C-terminal residues Arg of two antioxidative peptides might play an important role in scavenging free radical. These findings revealed the antioxidant mechanism of copra meal protein and provided theoretical support for its high-value utilization.

Identification of novel α-glucosidase and ACE inhibitory peptides from Douchi using peptidomics approach and molecular docking.

In this study, the effect of Douchi extract (DWE) on α-glucosidase and angiotensin-converting enzymes (ACE) were investigated, and several novel peptides with inhibitory activity against α-glucosidase and ACE were identified using peptidomics approach based on UPLC-MS/MS. The average inhibition rates of DWE on α-glucosidase and ACE were 73.75-78.10% and 4.56-27.07%, respectively. In the DWE, a total of 710 peptides were detected. Two novel peptides with potential inhibitory activity against α-glucosidase were identified using the correlation analysis, database alignment and molecular docking methods. They were DVFRAIPSEVL and DRPSINGLAGAN, with the IC50 values of 0.121 and 0.128mg/mL, respectively. Also, four novel peptides with potential inhibitory activity against ACE were identified: PSSPFTDLWD, EEQDERQFPF, PVPVPVQQAFPF and PSSPFTDL, with IC50 values of 1.388, 0.041, 0.761 and 0.097mg/mL, respectively. These results indicated that combining peptidomics and molecular docking is an effective alternative strategy for rapidly screening numbers of novel bioactive peptides from foods.

IAMY-RECMFF: Identifying amyloidgenic peptides by using residue pairwise energy content matrix and features fusion algorithm.

Various diseases, including Huntington's disease, Alzheimer's disease, and Parkinson's disease, have been reported to be linked to amyloid. Therefore, it is crucial to distinguish amyloid from non-amyloid proteins or peptides. While experimental approaches are typically preferred, they are costly and time-consuming. In this study, we have developed a machine learning framework called iAMY-RECMFF to discriminate amyloidgenic from non-amyloidgenic peptides. In our model, we first encoded the peptide sequences using the residue pairwise energy content matrix. We then utilized Pearson's correlation coefficient and distance correlation to extract useful information from this matrix. Additionally, we employed an improved similarity network fusion algorithm to integrate features from different perspectives. The Fisher approach was adopted to select the optimal feature subset. Finally, the selected features were inputted into a support vector machine for identifying amyloidgenic peptides. Experimental results demonstrate that our proposed method significantly improves the identification of amyloidgenic peptides compared to existing predictors. This suggests that our method may serve as a powerful tool in identifying amyloidgenic peptides. To facilitate academic use, the dataset and codes used in the current study are accessible at https://figshare.com/articles/online_resource/iAMY-RECMFF/22816916.

Identification and molecular interactions of novel ACE inhibitory peptides from rapeseed protein.

Plant-derived bioactive peptides have drawn much attention because of their physiological functions. This study aimed to evaluate bioactive peptides in rapeseed protein and identify novel angiotensin Ⅰ-converting enzyme (ACE) inhibitory peptides using bioinformatics methods. A total of 24 kinds of bioactive peptides were encrypted in the 12 selected rapeseed proteins by analysis in BIOPEP-UWM, with higher occurrence frequency of dipeptidyl peptidase Ⅳ (DPP-Ⅳ) inhibitory peptides (0.5727-0.7487) and ACE inhibitory peptides (0.3500-0.5364). Novel ACE inhibitory peptides FQW, FRW and CPF were identified by in silico proteolysis, and they had strong inhibitory effects on ACE in vitro, showing IC50 values of 44.84±1.48μM, 46.30±1.39μM and 131.35±3.87μM, respectively. Molecular docking results displayed that these three peptides were able to interact with ACE active site via hydrogen bonds and hydrophobic interactions, and coordinate with Zn2+. It suggested that rapeseed protein could be a good source for the production of ACE inhibitory peptides.

Preparation and identification of novel antioxidant peptides from camel bone protein.

Collagen hydrolysates are a vital source of bioactive peptides. The objective of this study was to prepare camel bone collagen hydrolysates with antioxidant activity, and to identify the peptides responsible for the antioxidant activity. To this end, single-factor and orthogonal tests were performed to explore the optimum preparation conditions. A hydrolysis time of 5h, enzyme:substrate ratio of 1200 U/g, pH of 7.0, and a material:water ratio of 1:3.0 were adopted. Subsequently, the hydrolysates were purified using a series of chromatography procedures, and three novel peptides, GPPGPPGPPGPPGPPSGGFDF (hydroxylation), PATGDLTDFLK, and GSPGPQGPPGSIGPQ, possessing antioxidant abilities, were identified from the fraction using liquid chromatography-tandem mass spectrometry. The peptide PATGDLTDFLK showed excellent DPPH scavenging activity (39%) and a good cytoprotective effect on H2O2-induced oxidative stress damage in HepG2 cells with a 21.1% increase observed.

Urine-HILIC: Automated Sample Preparation for Bottom-Up Urinary Proteome Profiling in Clinical Proteomics.

Urine provides a diverse source of information related to a patient's health status and is ideal for clinical proteomics due to its ease of collection. To date, most methods for the preparation of urine samples lack the throughput required to analyze large clinical cohorts. To this end, we developed a novel workflow, urine-HILIC (uHLC), based on an on-bead protein capture, clean-up, and digestion without the need for bottleneck processing steps such as protein precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample was subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using the novel approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analyzed by LCMS in data-independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. The data were searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein coefficient of variation was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥8.35-fold change in abundance, ≥2 unique peptides and ≤1% false discovery rate, and revealed 121 significant, differentially abundant proteins, some of which have known associations with kidney injury. The pilot data derived using this novel workflow provide information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.

Genome-Wide Identification of Trachinotus ovatus Antimicrobial Peptides and Their Immune Response against Two Pathogen Challenges.

Golden pompano, Trachinotus ovatus, as a highly nutritious commercially valuable marine fish, has become one of the preferred species for many fish farmers due to its rapid growth, wide adaptability, and ease of feeding and management. However, with the expansion of aquaculture scale, bacterial and parasitic diseases have also become major threats to the golden pompano industry. This study, based on comparative genomics, shows the possibility of preferential evolution of freshwater fish over marine fish by analyzing the phylogenetic relationships and divergence times of 14 marine fish and freshwater fish. Furthermore, we identified antimicrobial peptide genes from 14 species at the genomic level and found that the number of putative antimicrobial peptides may be related to species evolution. Subsequently, we classified the 341 identified AMPs from golden pompano into 38 categories based on the classification provided by the APD3. Among them, TCP represented the highest proportion, accounting for 23.2% of the total, followed by scolopendin, lectin, chemokine, BPTI, and histone-derived peptides. At the same time, the distribution of AMPs in chromosomes varied with type, and covariance analysis showed the frequency of its repeat events. Enrichment analysis and PPI indicated that AMP was mainly concentrated in pathways associated with disease immunity. In addition, our transcriptomic data measured the expression of putative AMPs of golden pompano in 12 normal tissues, as well as in the liver, spleen, and kidney infected with Streptococcus agalactiae and skin infected with Cryptocaryon irritans. As the infection with S. agalactiae and C. irritans progressed, we observed tissue specificity in the number and types of responsive AMPs. Positive selection of AMP genes may participate in the immune response through the MAPK signaling pathway. The genome-wide identification of antimicrobial peptides in the golden pompano provided a complete database of potential AMPs that can contribute to further understanding the immune mechanisms in pathogens. AMPs were expected to replace traditional antibiotics and be developed into targeted drugs against specific bacterial and parasitic pathogens for more precise and effective treatment to improve aquaculture production.