Search Results (233)

Marine invertebrates are a prime source of biologically active peptides due to their role in humoral immunity. These peptides typically exhibit broad-spectrum functions, including antibacterial, antifungal, anticancer, and immunomodulatory activities. In this report, we describe the identification and biological characterization of five novel bioactive peptides from the marine mollusk Pisania pusio. An extract of P. pusio was analyzed using nanoLC-ESI-MS-MS, and five peptides (PP1–5) were selected via bioinformatic screening as potential antimicrobial and anticancer peptides and subsequently validated experimentally. Among these, PP1, PP2, and PP4 were identified as cryptides derived from the proteolytic cleavage of actin, while PP3 and PP5 are novel peptides with no known protein precursors. All peptides exhibited moderate activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae with minimum inhibitory concentrations (MICs) predominantly at 100 µM. In contrast, only PP1 and PP5 were active against cancer cells, with PP1 being the most effective against A375 melanoma cells (IC₅₀ = 17.08 µM). This experimental validation confirmed the utility of the integrated in silico/peptidomic pipeline for lead identification. None of these peptides showed significant hemolytic activity or toxicity on fetal lung fibroblasts over 800 μM, demonstrating promising in vitro selectivity. These results highlight the multifunctional nature of P. pusio-derived peptides and their potential as lead compounds for further optimization and development into therapeutic agents against microbial infections and cancer, subject to more comprehensive safety evaluations in relevant models Full article

Type 2 diabetes mellitus (T2DM) is characterised by chronic hyperglycaemia and accumulation of advanced glycation end products (AGEs), driving interest in food-derived peptides as safer multifunctional modulators. Coconut milk is a promising source, but its anti-hyperglycaemic and anti-glycation potential remains largely unexplored. Here, proteins from coconut cream, skimmed and insoluble fractions of coconut milk were enzymatically hydrolysed, and the resulting peptides were profiled by nano-ESI-Orbitrap-LC-MS/MS. One hundred and fourteen peptides were identified and screened in silico against α-glucosidase, α-amylase, aldose reductase and the receptor for AGEs (RAGE). Two peptides, MQIFVK and ADVFNPR, showed the most favourable docking scores and physicochemical properties. However, ADVFNPR inhibited all 3 diabetic targets & RAGE. Molecular dynamics analysis showed that both peptides bind stably to the diabetic targets. Both peptides were synthesised and evaluated in vitro. ADVFNPR significantly inhibited α-glucosidase, α-amylase and aldose reductase with lower IC₅₀ values and displayed competitive inhibition kinetics. It also scavenged methylglyoxal, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and superoxide radicals at low EC₅₀ values, and showed low hemolytic activity in human erythrocytes. These findings indicate that coconut milk contains multifunctional peptides with anti-hyperglycaemic, anti-glycation and antioxidant activities that may be further developed as food-derived adjuncts for managing T2DM and glycation-related complications. Full article

Mini-LC systems, including Cap-LC, Nano-LC and Chip-LC, offer a sustainable alternative to conventional LC methods thanks to their reduced solvent consumption, enhanced separation efficiency and environmentally friendly operation. Integrating micro-scale sample preparation techniques, such as µ-SPE, IT-SPME, LPME and QuEChERS, with Mini-LC significantly improving analytical sensitivity and selectivity. Mini-LC coupled with mass spectrometry has demonstrated excellent performance in the detection of trace levels of pesticides, pharmaceuticals, veterinary drug residues, perfluoroalkyl substances (PFASs), and mycotoxins. Despite current challenges relating to matrix effects, instrument stability and method standardization, Mini-LC represents a promising analytical platform for the cost-effective, high-sensitivity, green monitoring of contaminants in food safety and environmental analysis. This review summarizes recent advances in the application of Mini-LC techniques for analyzing emerging organic contaminants (EOCs) in food and environmental samples. This paper also provides a critical review of this topic, covering works published in the last four years (early 2022–mid 2025). Additionally, it discusses the use of these techniques in combination with mass spectrometry (e.g., low-resolution MS or high-resolution MS) for the detection of EOCs in food and environmental samples. Full article

Background: Gram-positive bacteria, once considered incapable of producing extracellular vesicles (EVs) due to their thick peptidoglycan layer, are now known to secrete EVs that transport virulence factors and modulate host immunity. These EVs contribute to bacterial pathogenicity by facilitating biofilm formation, immune evasion, and inflammation. Granulicatella adiacens, an oral commensal associated with infective endocarditis, represents a clinically relevant model to study EV-mediated virulence. Objectives: This study’s aim was to investigate whether the proteomic composition and immunomodulatory activity of G. adiacens EVs differ between biofilm and planktonic lifestyles, thereby contributing to distinct pathogenic behaviours. Methods: EVs isolated from G. adiacens CCUG 27809 cultures were characterized using nano LC-ESI-MS/MS, followed by comprehensive bioinformatic and cytokine assays. Results: Quantitative proteomic profiling identified 1017 proteins, revealing distinct signatures between biofilm- and planktonic-derived EVs. Principal component analysis showed clear segregation between the two states, with biofilm EVs enriched in proteins linked to stress adaptation, adhesion, and structural integrity, while planktonic EVs exhibited growth- and metabolism-related proteins. A total of 114 virulence-associated proteins were identified, including several novel candidates. Functionally, EVs from both conditions significantly induced pro-inflammatory cytokines IL-8 and IL-1β in a dose-dependent manner (p < 0.05), whereas IL-17 remained unchanged. Conclusions: G. adiacens EVs exhibit lifestyle-dependent proteomic and immunomodulatory differences, underscoring their role in host–pathogen interactions and endocardial infection. These findings provide a foundation for future mechanistic and in vivo studies exploring EV-mediated virulence and potential therapeutic modulation. Full article

Several factors, including semen quality, can influence fertilisation success. Poor semen parameters may necessitate more frequent inseminations or the removal of males with consistently low fertility. This study evaluated turkey ejaculates (n = 37) with good fertility (GF) and impaired fertility (IF). The analyses included sperm motility parameters (total motility—TMOT, progressive motility—PMOT, curvilinear velocity—VCL, straight-line velocity—VSL, average path velocity—VAP, linearity—LIN, straightness—STR, amplitude of lateral head displacement—ALH, and beat cross frequency—BCF), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and nitric oxide (NO) production, as well as enzymatic and biochemical assays of semen, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) activities, glutathione (GSH) content, malondialdehyde (MDA) levels, and zinc (Zn²⁺) concentration. In parallel, the proteomes of seminal plasma and spermatozoa were separated using SDS- and Tricine-PAGE, and selected proteins were identified by nano LC-MS/MS. Spermatozoa derived from IF ejaculates exhibited significantly reduced TMOT (p = 0.002), VCL (p = 0.028), and PMI (p = 0.000), accompanied by elevated STR (p = 0.000) and NO production (p = 0.044). In the seminal plasma of IF males, a significant decrease was noted in SOD (p = 0.000) and GPx (p = 0.001) activities, whereas CAT activity was markedly higher (p = 0.014). Seminal fluid from IF ejaculates was also characterised by increased GSH (p = 0.014) and MDA (p = 0.014) concentrations, accompanied by reduced Zn²⁺ content (p = 0.014). In contrast, IF spermatozoa exhibited elevated SOD activity (p = 0.001), but reduced GPx (p = 0.000) and CAT (p = 0.012) activities. Sperm cells from IF ejaculates also had lower GSH levels (p = 0.000), higher MDA concentrations (p = 0.000), and increased Zn²⁺ content (p = 0.018) compared with those from GF ejaculates. A proteomic analysis revealed differences in fertility-associated proteins: peroxiredoxin 6 (PRDX6) was detected exclusively in GF semen, whereas alpha-enolase (ENO1), fatty acid-binding protein (FABP7), cytoplasmic aspartate aminotransferase (GOT1), and L-lactate dehydrogenase B (LDHB) were detected only in IF semen. Overall, the results demonstrate that both semen parameters and proteome composition may potentially affect the fertilisation outcomes in turkeys. Full article

Flap viability remains a major challenge in reconstructive surgery due to ischemia–reperfusion injury, excessive inflammation, and impaired tissue regeneration. Boron, a trace element with pro-healing and anti-inflammatory properties, has shown therapeutic promise in various wound models; however, its role in flap healing remains unclear. In this study, we aimed to evaluate the therapeutic potential of sodium pentaborate pentahydrate (SPP)-containing hydrogel, a boron compound we developed, for enhancing flap survival and tissue repair. A dorsal random-pattern flap model was established in male Wistar rats, which were treated topically with an SPP-containing formulation twice daily for seven days. Histological changes were evaluated using hematoxylin–eosin and Masson’s trichrome staining, and proteomic alterations were analyzed using label-free nanoLC-MS/MS followed by bioinformatics analysis. The treatment significantly improved flap survival (p < 0.0001), enhanced granulation tissue formation, promoted organized collagen deposition, and reduced inflammatory infiltration. Proteomic profiling identified 179 differentially expressed proteins, with 14 upregulated and 165 downregulated. Upregulated proteins were enriched in pathways related to complement activation, antioxidant defense, and extracellular matrix remodeling, whereas downregulated proteins were associated with immune overactivation, cellular stress, and senescence, indicating a shift toward regulated inflammation and tissue homeostasis. To our knowledge, this is the first study to demonstrate that an SPP-containing hydrogel promotes flap healing by supporting vascularization, modulating immune responses, and enhancing extracellular matrix remodeling. These findings highlight SPP as a promising therapeutic strategy for improving flap viability in reconstructive surgery. Full article

Extracellular vesicles (EVs) are bilayer-membrane cellular components that deliver protected cargo to the extracellular environment and can mediate long-distance signaling. We have previously reported that EVs isolated from the virulent fungal pathogen Paracoccidioides brasiliensis Vpb18 can revert the expression, in the attenuated variant Apb18, of stress-related virulence traits. We presently show that the Vev and Aev, respectively, produced by these variants display distinct proteomes, with prevalent functional enrichment in Vev related to oxidative stress response, signal transduction, transport, and localization, in addition to richer protein–protein interaction. Proteome sequences were obtained by nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-ESI-MS/MS). The Vev and corresponding Vpb18 proteomes also differed, suggesting a selective bias in vesicle protein cargo. Moreover, sublethal oxidative (VevOxi) and nitrosative (VevNO) stress modulated the Vev proteome and a positive correlation between VevOxi/VevNO-enriched and Vev-enriched (relative to Aev) proteins was observed. Out of 145 fungal virulence factors detected in Vev, 64% were enriched, strongly suggesting that molecules with virulence roles in Paracoccidioides are selectively concentrated in Vev. Our study significantly advanced the field by exploring protein N-terminal acetylation to a dimension rarely investigated in fungal EV proteomics. The proportion of N-terminally acetylated proteins in Vev was higher than in Vpb18 and the presence of Nt-acetylation in Vev-enriched virulence factors varied across the samples, suggesting that it might interfere with protein sorting into EVs and/or protein functionality. Our findings highlight the relevance of our fungal model to unraveling the significance of fungal EVs in pathogenesis and phenotypic transfer. Full article

The glyoxalase pathway intermediate S-d-lactoylglutathione was recently implicated in protein post-translational modifications in animal systems. Here, we examined the spontaneous modification of the Arabidopsis thaliana cytosolic glyceraldehyde-3-phosphate dehydrogenase C1 (GAPC1) by this compound. Incubation of GAPC1 with S-d-lactoylglutathione resulted in the inhibition of enzyme activity. The inhibitory effect was concentration dependent and increased at alkaline pHs. Furthermore, the inhibition of GAPC1 by S-d-lactoylglutathione was favored by oxidative conditions and reversed by reduction with dithiothreitol. Analyses of the S-d-lactoylglutathione-treated protein by nanoLC-MS/MS revealed S-glutathionylation of its two Cys residues and N-lactoylation of six Lys residues. Protein structure predictions showed that the double S-glutathionylation is accommodated by the GAPC1 catalytic pocket, which likely explains enzyme inhibition. N-lactoylated sites overlap partially with previously reported N-acetylated sites at the surface of the GAPC1 tetramer. The efficiency of cytosolic glutaredoxin and thioredoxin isoforms was tested for reversing the S-d-lactoylglutathione-induced modification. In these assays, recovery of GAPC1 activity after inhibition by S-d-lactoylglutathione treatment was used as indicator of efficiency. The results show that both types of redoxins were able to reverse inhibition. We propose a model describing the mechanisms involved in the two types of post-translational modifications found on GAPC1 following exposure to S-d-lactoylglutathione. The possible involvement of these findings for the control over glycolytic metabolism is discussed. Full article

The hydrocoral Millepora complanata (fire coral) plays a critical role in reef structure and relies on a symbiotic relationship with Symbiodiniaceae algae. Environmental stressors derived from climate change, such as UV radiation and elevated temperatures, disrupt this symbiosis, leading to bleaching and threatening reef survival. To gain insight into the thermal stress response of this reef-building hydrocoral, this study investigates the proteomic response of M. complanata to bleaching during the 2015–2016 El Niño event. Fragments from non-bleached and bleached colonies of the hydrocoral M. complanata were collected from a coral reef in the Mexican Caribbean, and proteomic extracts were analyzed using nano-liquid chromatography–tandem mass spectrometry (nano-LC-MS/MS). Uni- and multivariate analyses were applied to identify significant differences in protein abundance. A total of 52 proteins showed differential abundance, including 24 that showed increased expression and 28 whose expression decreased in bleached fragments. Differentially abundant proteins were associated with amino acid biosynthesis, carbohydrate metabolism, cytoskeleton organization, DNA repair, extracellular matrix composition, redox homeostasis, and protein modification. These molecular alterations reflect critical physiological adaptations that may influence stress sensitivity or tolerance in hydrocorals. The findings indicate that heat stress induces molecular responses involving protein refolding, enhanced vesicular transport, cytoskeletal reorganization, and modulation of redox activity. This contributes to a deeper understanding of the molecular mechanisms underlying bleaching in reef-building hydrozoans and broadens current knowledge beyond the more extensively studied anthozoan corals. Full article

Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and cellular signaling in cancer. Their functions are primarily mediated through interactions with specific protein partners that modulate chromatin structure, epigenetic remodeling, transcription, and signal transduction. In this review, we explore reports and strategies for the proteomic characterization of lncRNA-associated proteins, particularly emphasizing high-throughput liquid chromatography–mass spectrometry (LC-MS)-based techniques. Affinity-based methods such as RNA pull-down, ChIRP MS, RAP-MS, BioID-MS, and SILAC-MS enable sensitive and specific mapping of lncRNA and protein complexes. These approaches reveal cancer-specific proteomic signatures, post-translational modifications, and mechanistic insights into tumor biology. The use of label-free quantification, bituminization, and crosslinking strategies further enhances the resolution of dynamic RNA–protein networks. Validation tools following bioinformatic analyses, such as Western blotting, immunohistochemistry, immunofluorescence, and ELISA, are used to prioritize and confirm findings. Candidate biomarkers from hepatocellular carcinoma to colorectal and prostate cancers, profiling lncRNA-associated proteins, hold promise for identifying clinically actionable biomarkers and therapeutic targets. This review highlights the translational relevance of lncRNA protein studies and advocates for their broader adoption in oncological research. In LC-MS workflows, proteins bound to lncRNAs are enzymatically digested into peptides, separated via nano-LC, and analyzed using high-resolution tandem MS. Label-free or isotope-labeled methods quantify differential enrichment, followed by bioinformatics-driven pathway annotation. Full article

Although Hylocereus polyrhizus pulp residues polysaccharides (HPPP) have shown potential in improving metabolic disorders and intestinal barrier function, the mechanism by which they exert their effects through regulating O-glycosylation modifications in the mucus layer remains unclear. Therefore, this study established a HFD-induced obese colitis mouse model (n = 5 per group) and combined nano-capillary liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) technology to quantitatively analyze the dynamic changes in O-glycosylation. Additionally, through quantitative O-glycosylation proteomics and whole-proteome analysis, we identified 155 specifically altered O-glycosylation sites in colon tissue, with the glycosylation modification level of the MUC2 core protein increased by approximately 2.1-fold. The results indicate that HPPP alleviates colonic mucosal damage by regulating interactions between mucus O-glycosylation. Overall, we demonstrated that HPPP increases HFD-induced O-glycosylation sites, improves intestinal mucosal structure in obese mice, and provides protective effects against obesity-induced intestinal mucosal damage. Full article

Xerostomia, the subjective complaint of a dry mouth, is frequently associated with salivary flow reduction and/or salivary gland hypofunction. This condition significantly impacts an individual’s quality of life and oral health, including difficulties in speaking, chewing, and swallowing. Xerostomia may be caused by autoimmune diseases, xerogenic medications, and radiation therapy. Our objective was to identify differentially expressed proteins in the saliva of patients with medication and autoimmune disease-associated xerostomia compared to non-xerostomic control subjects. Two groups of individuals (N = 45 total) were recruited: non-xerostomic subjects (NX-group; n = 18) and xerostomic patients (XP-group; n = 27). Dried saliva spot samples were collected from major salivary glands, i.e., parotid (left and right) and submandibular glands. Proteomic analysis was performed by deep nanoLC-MS/MS. Differential protein expression in the XP-group relative to the NX-group was determined by the Mann–Whitney U-test with FDR Benjamini–Hochberg correction (p_adj < 0.05). The Search Tool for Recurring Instances of Neighboring Genes (STRING_v12.0) was used to generate interaction networks and perform pathway analysis. A total of 1407 proteins were detected. Of these, 86 from the left parotid gland, 112 from the right parotid gland, and 73 from the submandibular gland were differentially expressed proteins (DEPs). Using STRING analysis, we identified, for the first time, several neurodegenerative disease-associated networks, primarily involving the downregulation of the 20S proteasome core complex and glyoxalase proteins across salivary glands. In this study, we determined neuronal dysregulation and impaired methylglyoxal (MGO) detoxification, possibly through reduced protein expression of glyoxalase Parkinson’s Disease (PD) Protein 7 (encoded by the PARK7 gene) in major salivary glands of xerostomic patients. Indeed, impaired MGO detoxification has been previously shown to cause salivary gland dysfunction in a mouse model of type 2 diabetes. Based on other DEPs associated with neurodegenerative disorders, our results also suggest a possible deficiency in the parasympathetic nervous system innervation of salivary glands, warranting further investigation. Full article

Nano-titanium dioxide (nano-TiO₂) can alleviate oxidative damage in plants subjected to abiotic stress, interfere with related gene expression, and change metabolite content. Polylactic acid (PLA) microplastics can inhibit plant growth, induce oxidative stress in plant cells, and alter the biophysical properties of rhizosphere soil. In this study, untargeted metabolomics (LC-MS) and RNA-seq sequencing were performed on radish root cells exposed to nano-TiO₂ and PLA. The results showed that nano-TiO₂ alleviated the growth inhibition of radish roots induced by PLA. Nano-TiO₂ alleviated PLA-induced oxidative stress, and the activities of SOD and POD were decreased by 28.6% and 36.0%, respectively. A total of 1673 differentially expressed genes (DEGs, 844 upregulated genes, and 829 downregulated genes) were detected by transcriptome analysis. Metabolomics analysis showed that 5041 differential metabolites were involved; they mainly include terpenoids, fatty acids, alkaloids, shikimic acid, and phenylpropionic acid. Among them, phenylpropanoid biosynthesis as well as flavone and flavonol biosynthesis were the key metabolic pathways. This study demonstrates that nano-TiO₂ mitigates PLA phytotoxicity in radish via transcriptional and metabolic reprogramming of phenylpropanoid biosynthesis. These findings provide important references for enhancing crop resilience against pollutants and underscore the need for ecological risk assessment of co-existing novel pollutants in agriculture. Full article

Tonsils are secondary lymphoid organs that play a crucial role in the immunological response, with B cells being a major component involved in both innate and adaptive immunity. Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome and obstructive sleep apnea syndrome (OSAS) are both common pediatric conditions involving tonsillar pathology. In both syndromes, the molecular pathways dysregulated in tonsillar B cells are still to be understood. The study aimed to unravel and compare the proteomic profiles of tonsillar CD19+ B cells isolated from pediatric patients with PFAPA (n = 6) and OSAS (n = 6) to identify disease-specific molecular signatures. B cells were isolated from the tonsillar tissue using magnetic microbeads (with a purity of 93.50%). Proteomic analysis was performed by nanoLC-MS/MS with both data-dependent (DDA) and data-independent acquisition (DIA) methods, followed by comprehensive bioinformatic analysis. By merging DDA and DIA datasets, a total of 18.078 unique proteins were identified. Differential expression analysis revealed 83 proteins increased and 49 proteins decreased in OSAS B cells compared to PFAPA B cells (fold change ≥ 1.5 or ≤0.6, p < 0.05). Distinct pathway enrichments were highlighted, including alterations in the regulation of PTEN gene transcription, circadian gene expression, inflammasome pathways (IPAF and AIM2), and the metabolism of angiotensinogen to angiotensin. Specific proteins such as p53, Hdac3, RPTOR, MED1, Caspase-1, Cathepsin D, Chymase, and TLR2 (validated by WB) were shown to be differentially expressed. These findings reveal distinct proteomic signatures in tonsillar B cells from patients with PFAPA and OSAS, offering novel insights into the pathophysiology and potential avenues for biomarker discovery. Full article

Springtails are adapted to life in the pore space of soil, where humidity in moist soil is close to saturation. Drought is the most important limiting factor for springtails; however, their molecular and physiological adaptations to low humidity are not well understood. The present study explored the global proteomic drought response of the springtail, Folsomia candida (Isotomidae, Collembola). In relatively dry soil (−360 kPa), adult springtails initially lost body water but re-established the normal body water content over the following two weeks. Nano LC–MS/MS analysis identified a total of 1729 unique proteins. Proteomic analysis and pathway enrichment found that the proteome generally did not show a dramatic induction of proteins in response to drought stress. After an initial down-regulation of pathways related to metabolism and growth, these pathways gradually returned to the same levels as in moist soil. Other pathways such as the cytoskeleton pathway, which is important in cell proliferation and differentiation, were predominantly down-regulated throughout the experiment in drought-exposed animals, which correlated with essentially no somatic growth of the springtails in dry soil. This study facilitates the understanding of the consequences of climate change on soil functioning and fertility. Full article