Search Results (128)

Although antiviral therapy has improved quality of life, around 50% of people with HIV (PWH) experience neurodegeneration and cognitive decline. This is prompted in part by the migration of HIV-infected monocyte-derived macrophages (MDMs) to the brain, leading to neuronal death. Previous studies in our lab have shown that HIV-infected MDMs secrete cathepsin B (CATB), which is a pro-inflammatory neurotoxic enzyme that is reduced by the addition of cannabinoid receptor-2 (CB2R) agonist JWH-133 to cell cultures. In this study, we aimed to identify the proteins secreted (secretome) by HIV-infected macrophages exposed to JWH-133 and quantify them using tandem mass tag (TMT) mass spectrometry. Frozen 13-day MDM supernatants from (1) an MDM negative control; (2) HIV+MDM, and (3) HIV+MDM-JWH-133 were compared in triplicate by mass spectrometry (LC/MS/MS) and analyzed for protein identification. Subsequently, the same samples were labeled by TMT labeling and quantified by LC/MS/MS. After a database search, 528 proteins were identified from all groups. Thereafter, proteins with more than three unique peptides and more than 10% coverage were selected for protein identification. Venn diagrams revealed one unique protein secreted by MDM-HIV, 10 unique proteins in HIV+MDM-JWH-133, and 15 common proteins in the three groups. CATB was unique to HIV+MDM. HIV+MDM exposed to JWH-133 showed proteins related to metabolism, cell organization, antiviral activity, and stress response. TMT analysis revealed 1454 proteins with abundance for statistical analysis based on FC ≥ |1.5| and p-value ≤ 0.05, of which Ruvb-like 1 and Hornerin decreased significantly with JWH-133 treatment. Both proteins stimulate HIV replication. In addition, HIV infection upregulated proteins associated with pathways of viral latency that were inhibited by JWH-133. In conclusion, JWH-133 treatment in HIV-infected macrophages leads to the secretion of antiviral host factors and decreases the secretion of proviral, inflammatory, and neurotoxic host factors. Full article

Background/Objectives: Disruption of AMPAR trafficking at excitatory synapses contributes to impaired synaptic plasticity and memory formation in several neurological and psychiatric disorders. Arc, an immediate early gene product, has been shown to interact with the AMPAR auxiliary subunit TARPγ2, affecting receptor mobility and synaptic stabilization. Methods: To investigate the in vivo functional effects and protein interactions of the Arc-TARPγ2 interfering peptide RIPSYR, we performed in vivo electrophysiology and spatial memory assessments in male rats. as well as proteomic analyses of peptide-protein interactions in synaptosome lysates. We then used in silico docking to evaluate candidate binding partners. Results: In the present study, in vivo electrophysiological measurements revealed that RIPSYR administration altered early-phase long-term potentiation at CA3 synapses of male rats. Subsequent behavioral testing that assessed spatial memory performance revealed depleted memory retrieval after 24 h, indicating that the peptide has a systemic effect on experience-dependent plasticity. Then, we examined the molecular interactome of RIPSYR using magnetic bead-based immunoprecipitation and subsequent LC-MS identification on synaptosome lysates, and identified additional candidate binding partners, suggesting that the peptide may have broader modulatory effects. RIPSYR binding to the other putative binding partners are investigated by in silico methods. Conclusion: Our results raise the question of how the molecular interactions of RIPSYR contribute to its sum effects on electrophysiology and behavior. Full article

Acanthophis antarcticus, commonly known as the death adder, is a venomous Australian snake and a member of the Elapidae family. Due to its robust body and triangular head, it was historically misclassified as a viper. Its venom is known for neurotoxic, hemorrhagic, and hemolytic effects but displays low anticoagulant activity. Although key toxins such as three-finger toxins (3FTxs) and phospholipase A₂ (PLA₂) have been previously described, no study has integrated proteomic and functional analyses to date. In this study, we conducted a comprehensive characterization of A. antarcticus venom. Reverse-phase high-performance liquid chromatography (RP-HPLC) followed by LC-MS/MS enabled the identification of nine toxin families, with 3FTxs and PLA₂ as the most abundant. Less abundant but functionally relevant toxins included Kunitz-type inhibitors, CRISP, SVMP, LAAO, NGF, natriuretic peptides, and nucleotidases, the latter being reported here for the first time based on proteomic evidence. Hydrophilic interaction chromatography (HILIC) coupled with MALDI-TOF was used to analyze polar, non-retained venom components, revealing the presence of low-molecular-weight peptides (2–4 kDa). Functional assays confirmed the enzymatic activity of HYAL, PLA₂, and LAAO and, for the first time, demonstrated inhibitory activity on serine peptidases and fibrinogenolytic activity in the venom of this species. These findings expand our understanding of the biochemical and functional diversity of this venom. Full article

Mussels are nutrient-rich but perishable, resulting in substantial resource loss. A protease-producing strain (Bacillus velezensis Z-1, Mytilus edulis) isolated from marine sludge was used to hydrolyze mussels, producing Y-1, a hydrolysate with antioxidant activity. In this study, ultrafiltration, gel chromatography, and LC-MS/MS were employed to isolate and identify bioactive peptides from the hydrolysate. The results revealed that the hydrolysate exhibited antioxidant activity, pancreatic cholesterol esterase inhibitory activity, pancreatic lipase inhibitory activity, and α-glucosidase inhibitory activity. Molecular docking using AutoDock Tools 1.5.6 was performed to analyze the interactions of peptides with CD38 and Keap1, leading to the identification of five potentially bioactive peptides: VPPFY, IMLFP, LPFLF, FLPF, and FPRIM. These peptides formed hydrogen bonds and hydrophobic interactions with CD38 and Keap1, demonstrating strong DPPH radical scavenging and superoxide anion radical scavenging capacities. This study highlights the multifunctional bioactive potential of these peptides, offering insights into their therapeutic applications. The findings provide a novel approach for the effective utilization of mussel resources and highlight their potential application value in the development of functional foods. Full article

This review highlights the critical role of chemotaxonomy in the identification, authentication, and discovery of bioactive compounds in medicinal plants. By analyzing secondary metabolites using techniques like UV spectroscopy, FTIR, HPLC, GC-MS, NMR, LC-MS-Qtof, and MALDI-TOF MS, chemotaxonomy ensures accurate plant identification, supporting the safe and effective use of plants in herbal medicine. Key secondary metabolites used in chemotaxonomic identification include alkaloids, flavonoids, terpenoids, phenolics, tannins, and plant peptides. Chemotaxonomy also facilitates the discovery of novel compounds with therapeutic potential, contributing to drug development. The integration of chemotaxonomy with genomics and proteomics allows a deeper understanding of plant biosynthesis and the mechanisms behind bioactive compound production. However, challenges due to variability in metabolite profiles and the lack of standardized methods remain, and future research should focus on developing global databases, improving standardization, and incorporating artificial intelligence and machine learning to enhance plant identification and bioactive compound discovery. The integration of chemotaxonomy with personalized medicine offers the potential to tailor plant-based therapies to individual genetic profiles, advancing targeted treatments. This review underscores chemotaxonomy’s importance in bridging traditional knowledge and modern science, offering sustainable solutions for medicinal plant use and drug development. Full article

Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or non-PDO buffalo milk in PDO dairy buffalo products. Peripheral laboratories use gel electrophoresis combined with polyclonal antipeptide antibodies for initial screening, enabling the detection of foreign caseins, including those originating outside the PDO-designated regions. For more precise identification, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) differentiates species by detecting proteotypic peptides. In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. On the other hand, analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration. Despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection, providing a robust tool to face increasingly sophisticated fraudulent practices. Full article

Bioactive peptides derived from milk proteins offer promising potential that can be unlocked through hydrolysis. Enzymatic hydrolysis is particularly noteworthy because of its mild conditions and its efficacy in producing peptides with various biological activities. This study focused on creating whey protein hydrolysates using three enzymes: pepsin, trypsin, and papain. The degree of hydrolysis and the antioxidant properties of the resulting peptides were evaluated, and papain demonstrated the highest degree of hydrolysis, leading to its selection for further investigation. LC-MS was employed to identify peptide sequences from the papain-derived hydrolysate, resulting in the identification of 107 distinct peptide sequences These peptides were predicted to exhibit a range of potential biological activities, including antihypertensive, antidiabetic, antioxidant, antimicrobial, and immunomodulatory effects, as well as roles in regulating glucose homeostasis, maintaining cardiovascular health, and supporting overall metabolic function. In vitro tests revealed the significant antioxidant and antibacterial properties of the hydrolysate, confirming the potential of papain-derived peptides for use in functional food and pharmaceutical applications. The novelty of this study lies in the identification of novel peptides with promising biological activities. Additional in vitro and in vivo studies are required to fully elucidate the health benefits of these peptides. Full article

Background/Objectives: Lactic acid bacteria (LAB) produce several diverse metabolites during fermentation that play key roles in enhancing health and food quality. These metabolites include peptides, organic acids, exopolysaccharides, and antimicrobial compounds, which contribute to gut health, immune system modulation, and pathogen inhibition. This study analyzed the intracellular (Met-Int) and extracellular metabolites (Met-Ext-CFS; cell-free supernatant) of Lactiplantibacillus plantarum UTNGt2, a probiotic strain isolated from Theobroma grandiflorum. Methods: The assessment was performed using capillary LC-MS/MS metabolomics with a SWATH-based data-independent acquisition approach to identify molecules associated with antimicrobial activity. Results: The integration of metabolomic data with whole-genome annotation enabled the identification of several key metabolites, including amino acids, nucleotides, organic acids, oligopeptides, terpenes, and flavonoids, many of which were associated with the antimicrobial activity of UTNGt2. Pathway analysis reveals critical processes such as secondary metabolite biosynthesis, nucleotide and galactose metabolism, and cofactor biosynthesis. By integrating RiPP (ribosomally synthesized and post-translationally modified peptide) cluster gene predictions with LC-MS data, this study validates the production of specific RiPPs and uncovers novel bioactive compounds encoded within the UTNGt2 genome. The oligopeptide val-leu-pro-val-pro-gln found in both Met-Int (ESI+) and Met-Ext-CFS (ESI+) may contribute to the strain’s antimicrobial strength. It could also enhance probiotic and fermentation-related functions. Conclusions: While genome-based predictions highlight the strain’s biosynthetic potential, the actual metabolite profile is influenced by factors like transcriptional regulation, post-transcriptional and post-translational modifications, and environmental conditions. These findings emphasize the value of multi-omics approaches in providing a holistic understanding of metabolite production and its role in antimicrobial activity. Full article

Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are considered the most important mycotoxins in terms of food safety. The aim of this study was to evaluate the hepatotoxicity of AFB1 and OTA exposure in Wistar rats and to assess the beneficial effect of fermented whey (FW) and pumpkin (P) as functional ingredients through a proteomic approach. For the experimental procedures, rats were fed AFB1 and OTA individually or in combination, with the addition of FW or a FW-P mixture during 28 days. For proteomics analysis, peptides were separated using a LC-MS/MS-QTOF system and differentially expressed proteins (DEPs) were statistically filtered (p < 0.05) distinguishing males from females. Gene ontology visualization allowed the identification of proteins involved in important biological processes such as the response to xenobiotic stimuli and liver development. Likewise, KEGG pathway analysis reported the metabolic routes as the most affected, followed by carbon metabolism and biosynthesis of amino acids. Overall, the results highlighted a strong downregulation of DEPs in the presence of AFB1 and OTA individually but not with the mixture of both, suggesting a synergistic effect. However, FW and P have helped in the mitigation of processes triggered by mycotoxins. Full article

Invertebrate tropomyosins belong to the key food allergens. Several peptides likely to be released during proteolysis can be found in many sequences of proteins from this family. The aim of the present study was to evaluate the possibility of identifying tropomyosins with known and unknown amino acid sequences in unheated, boiled and fried seafoods. The workflow included in silico proteolysis simulation of tropomyosin sequences and analysis of the distribution of resulting peptides among proteins. The experiment entailed the proteolysis of unheated, boiled and fried products, containing crustaceans or mollusks, and the identification of resulting peptides using LC-MS/MS. Finally, taxonomic lineages of identified peptides were determined. Predicted peptides were identified in unheated samples. The boiling of seafoods resulted in an increase in the length of peptides containing predicted sequences. Some peptides from the boiled samples contained entire linear epitopes. The prediction of tropomyosin cleavage sites failed in the case of fried products. Peptides from the unheated and boiled samples were attributed to crustacean, arthropod or molluscan tropomyosins. In turn, peptides from the fried samples possessed inconclusive taxonomic lineages. Our results show that bioinformatics analysis (especially using Unipept program) may be a viable tool supporting LC-MS/MS experiments aimed at the detection of allergens. Full article

Tick eggs contain a series of proteins that play important roles in egg development. A thorough characterization of egg protein expression throughout development is essential for understanding tick embryogenesis and for screening candidate molecules to develop novel interventions. In this study, eggs at four developmental stages (0, 7, 14, and 21 incubation days) were collected, and their protein extraction was profiled using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). On the first day of egg protein extraction, protein bands from day-1 eggs were re-collected and subsequently analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The dynamic changes in forty egg proteins during development were further investigated using LC-parallel reaction monitoring (PRM)/MS analysis. A total of 108 transcripts were detected in day-1 eggs. Based on protein functions and families, these transcripts were classified into eight categories: transporters, enzymes, immunity and antimicrobial proteins, proteinase inhibitors, cytoskeletal proteins, heat shock proteins, secreted proteins, and uncharacterized proteins. Identification of the protein bands revealed that nine bands predominantly consisted of vitellogenin and vitellin-A, while other notable proteins included cathepsins and Kunitz domain-containing proteins. LC-PRM/MS analysis indicated that 28 transcripts increased significantly in abundance, including 13/18 enzymes, 1/1 antimicrobial peptide, 2/2 neutrophil elastase inhibitors, 3/4 vitellogenins, 3/3 heat shock proteins, 3/3 cytoskeletal proteins, 1/1 elongation factor-1, and 1/1 uncharacterized protein. Conversely, five transcripts showed a decrease significantly, including 1/1 Kunitz domain-containing protein, 2/6 aspartic proteases, and 2/5 serpins. This research provides a comprehensive overview of egg proteins and highlights the dynamic changes in protein expression during embryonic development, which may be pivotal for understanding protein functions and selecting potential candidates for further study. Full article

Antimicrobial resistance (AMR) poses a significant challenge to animal production due to the widespread use of antibiotics. Therefore, there is an urgent need for alternative antimicrobial strategies to effectively manage bacterial infections, protect animal health, and reduce reliance on antibiotics. This study evaluated the use of emerging approaches and procedures for the isolation, identification, and characterization of bacteriocin-producing bacteria and their bacteriocins, sourced from the gastrointestinal tract (GIT) of meat-producing pigs. Out of 2056 isolates screened against Gram-positive and Gram-negative indicator strains, 20 of the most active antimicrobial isolates were subjected to whole genome sequencing (WGS) for the prediction of coding DNA sequences (CDS) and the identification of bacteriocin gene clusters (BGC) and their functions. The use of an in vitro cell-free protein synthesis (IV-CFPS) protocol and the design of an IV-CFPS coupled to a split-intein mediated ligation (IV-CFPS/SIML) procedure made possible the evaluation of the production and antimicrobial activity of described and putatively novel bacteriocins. A colony MALDI-TOF MS procedure assisted in the identification of class I, II, and III lanthipeptides. MALDI-TOF MS and a targeted proteomics, combined with a massive peptide analysis (LC-MS/MS) approach, has proven valuable for the identification and biochemical characterization of previously described and novel bacteriocins encoded by the isolated bacteriocin-producing strains. Full article

Sacha Inchi (Plukenetia volubilis) oil press-cake (SIPC) represents a new source of proteins of high biological value, with promissory food applications. However, knowledge of these proteins remains limited. In this study, a Sacha Inchi protein concentrate (SPC) was extracted from the SIPC, and proteomic analysis was performed to identify the major alkaline-soluble proteins. The electrophoretic profile highlighted the efficacy of alkaline pH and moderate temperature to extract the major proteins, from which a group of proteins, not previously reported, were registered. LC-MS/MS analyses produced abundant high-quality fragmentation spectra. Utilizing the Euphorbiaceae database (DB), 226 proteins were identified, with numerous well-assigned spectra remaining unidentified. PEAKS Studio v11.5 software generated 1819 high-quality de novo peptides. Data are available via ProteomeXchange with identifier PXD052665. Gene ontology (GO) classification allowed the identification of sequenced proteins associated with biological processes, molecular functions, and cellular components in the seed. Consequently, the principal alkali-soluble proteins from SPC were characterized through derived functional analysis, covering 24 seed-storage-, 27 defense-, and 12 carbohydrate- and lipid-metabolism-related proteins, crucial for human nutrition due to their sulfur-containing amino acids, antioxidant properties, and oil yields, respectively. This research makes a significant contribution to the current understanding of the Sacha Inchi proteome and offers valuable insights for its potential applications in the food industry. Full article

Identification of the individuals having impaired kidney function is essential in preventing the complications of this disease. We measured 1009 metabolites at the baseline study in 10,159 Finnish men of the METSIM cohort and associated the metabolites with an estimated glomerular filtration rate (eGFR). A total of 7090 men participated in the 12-year follow-up study. Non-targeted metabolomics profiling was performed at Metabolon, Inc. (Morrisville, NC, USA) on EDTA plasma samples obtained after overnight fasting. We applied liquid chromatography mass spectrometry (LC-MS/MS) to identify the metabolites (the Metabolon DiscoveryHD4 platform). We performed association analyses between the eGFR and metabolites using linear regression adjusted for confounding factors. We found 108 metabolites significantly associated with a decrease in eGFR, and 28 of them were novel, including 12 amino acids, 8 xenobiotics, 5 lipids, 1 nucleotide, 1 peptide, and 1 partially characterized molecule. The most significant associations were with five amino acids, N-acetylmethionine, N-acetylvaline, gamma-carboxyglutamate, 3-methylglutaryl-carnitine, and pro-line. We identified 28 novel metabolites associated with decreased eGFR in the 12-year follow-up study of the METSIM cohort. These findings provide novel insights into the role of metabolites and metabolic pathways involved in the decline of kidney function. Full article

Immunopeptidomics is the area of knowledge focused on the study of peptides assembled in the major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, which could activate the immune response via specific and selective T cell recognition. Advances in high-sensitivity mass spectrometry have enabled the detailed identification and quantification of the immunopeptidome, significantly impacting fields like oncology, infections, and autoimmune diseases. Current immunopeptidomics approaches primarily focus on workflows to identify immunopeptides from HLA molecules, requiring the isolation of the HLA from relevant cells or tissues. Common critical steps in these workflows, such as cell lysis, HLA immunoenrichment, and peptide isolation, significantly influence outcomes. A systematic evaluation of these steps led to the creation of an ‘Immunopeptidome Score’ to enhance the reproducibility and robustness of these workflows. This score, derived from LC-MS/MS datasets (ProteomeXchange identifier PXD038165), in combination with available information from public databases, aids in optimizing the immunopeptidome characterization process. The ‘Immunopeptidome Score’ has been applied in a systematic analysis of protein extraction, HLA immunoprecipitation, and peptide recovery yields across several tumor cell lines enabling the selection of peptides with optimal features and, therefore, the identification of potential biomarker and therapeutic targets. Full article