Goat blood, a major slaughterhouse by-product, was systematically valorized into dual-function bioactive peptides through an optimized four-step process. Four blood preparations-whole blood (HB), anticoagulant-treated blood (HBS), red blood corpuscles (BC), and plasma (PM)-were subjected to heat pretreatment (90 °C, 15 min) and enzymatic hydrolysis. Neutrase hydrolysis of heat-pretreated whole blood at 8% substrate concentration for 4 h (HBN-8) yielded optimal protein recovery (44.38%) with dual ACE (88.24%) and DPP-IV (81.13%) inhibition. Ultrafiltration enriched bioactive peptides in the ≤3 kDa fraction (DPP-IV: 87.8%; ACE: 65.5%). LC-MS/MS de novo sequencing identified 14 novel peptide sequences (4-9 amino acids), with the most potent SEC fraction showing IC50 values of 0.89 and 0.45 mg Leu eq./mL for DPP-IV and ACE inhibition, respectively. Critically, simulated gastrointestinal digestion enhanced rather than diminished bioactivity, with ACE inhibition increasing progressively to 60.91% at the intestinal phase, supported by predicted generation of bioactive fragments from parent sequences. Caco-2 assays confirmed peptide safety (100-1000 µg/mL) and demonstrated 10.47% transepithelial transport with retained dual inhibitory activities. This study establishes goat blood as a sustainable source of orally bioavailable, GI-stable peptides for the development of functional foods targeting hypertension and type 2 diabetes.

Aging, a complex biological process, is intrinsically linked to the pathogenesis of numerous age-related diseases. A key factor in the aging process is the accumulation of DNA damage and the subsequent activation or failure of the DNA damage response. To mitigate this damage, DNA repair mechanisms often involve the formation of DNA gaps. This study investigates the potential role of the Box A domain of High Mobility Group Box 1 (HMGB1) in modulating age-related changes. We utilized a label-free quantitative proteomic technique to analyze the plasma proteome of three female adult and eight female perimenopausal cynomolgus macaques (Macaca fascicularis), with the perimenopausal group receiving an intravenous administration of the Box A plasmid. Proteomic analysis revealed differential expressions in proteins primarily associated with stress response, immune regulation, lipid transport, and cellular homeostasis following Box A plasmid intervention. Notably, the expression levels of key proteins, such as apolipoprotein E (APOE) and sex hormone-binding globulin (SHBG), showed a reversal effect, restoring levels closer to those observed in the younger, adult monkeys. These findings highlight the potential of the Box A of HMGB1 plasmid as a therapeutic candidate to mitigate age-related proteomic alterations, offering a novel avenue for targeted interventions in aging and associated diseases.

Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer-related death worldwide. Early detection can reduce CRC mortality by more than 90%. Circulating small extracellular vesicles (sEVs) are emerging as promising biomarkers for CRC, but their role in detecting precancerous lesions remains unclear. Herein, parallel proteomic and phosphoproteomic analyses of plasma-derived sEVs were performed in healthy subjects with negative colonoscopy, patients with high-risk adenoma (HRA) and patients with CRC. A total of 139 phosphorylation sites on 52 proteins were identified, among which 16 phosphorylation sites on 12 sEV proteins showed significant changes (≥ 2-fold) with 90% confidence in site localization. Web-based validation demonstrated that the phosphorylation level of sEV-derived filamin-A at serine 1459 (pFLNASer1459) correlated with the Clinical Proteomic Tumor Analysis Consortium colon cancer dataset. Immunoblot analysis confirmed that sEV-derived pFLNASer1459 was significantly reduced in CRC patients compared with healthy subjects, whereas the highest levels were observed in HRA patients. Notably, sEV-derived pFLNASer1459, alone or in combination with FLNA, CD9, and TSG101, showed superior diagnostic performance in distinguishing HRA patients from CRC patients and healthy subjects. These findings suggest that plasma sEV-derived pFLNASer1459 is a promising biomarker for colorectal neoplasm detection.

Cowpea (Vigna unguiculata L. Walp) seeds are rich in proteins (∼24%), thus they have been considered as a viable dietary protein substitute. Despite its advantages, allergenicity risks of cowpea seeds need to be taken into account. Herein, the protein expression and potential allergens in cowpea seeds were analyzed using LC/timsTOF Pro 2, PEAK studio and multiple in silico analysis. Based on functional classification using STRING analysis, the result revealed response to stimuli, e.g., oxidative stress and temperature, as the major cluster (187 proteins), followed by biosynthesis of secondary metabolite (117 proteins) and immune system (108 proteins). These suggest involvement of protein functions in maintaining homeostasis during seed development under stress conditions. By using webtool Allermatch™ and the Pfam database, 131 potential allergenic proteins were found from cowpea seeds. The findings revealed that cowpea seeds contain a number of recognized allergens, including endochitinase, beta-conglycinin, and vicilin, as well as numerous allergenic proteins not previously described, such as endochitinase 1B and 5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase (MetE). Additionally, use of the Kolaskar & Tongaonkar method predicted B-cell epitopes such as 30VSGFGVI36, 152VPVLVGP158 and 153PVLVGPV159, increasing the possibility of cowpea allergenicity. In conclusion, this study provides useful information on the potential allergens in cowpea seeds, providing a foundation for future cowpea allergenicity assessment including experimental IgE-binding or clinical validation.

O-GlcNAcylation, a single attachment of N-acetylglucosamine (GlcNAc) on serine/threonine residues of nuclear-cytoplasmic proteins, is frequently upregulated in various cancers and implicated in several aspects of tumor progression. Growing evidence reports that treatments of chemotherapeutic drugs may activate protein O-GlcNAcylation. However, its precise role in modulating chemotherapeutic responses, particularly in colorectal cancer (CRC), remains poorly defined. Herein, we investigate the biological effects of oxaliplatin (OXA), a first-line chemotherapy drug for patients with metastatic CRC, and protein O-GlcNAcylation reduction in SW620 metastatic CRC cells. OXA treatment alone reduced cell viability as well as proliferation, and increased the levels of protein O-GlcNAcylation and GFPT1, the rate limiting enzyme of hexosamine biosynthetic pathway which is a nutrient sensor of glucose metabolism. Inhibition of protein O-GlcNAcylation via genetic knockdown of O-GlcNAc transferase (OGT) or chemical inhibition (OSMI-1) markedly enhanced SW620 sensitive to OXA. This was evidenced by decreased cell viability and proliferation, increased cell apoptosis, and cell cycle arrest. Mass spectrometry-based proteomics and bioinformatics analysis revealed that the combination of OGT knockdown and OXA treatment majorly downregulated several ribosomal proteins. In addition, OXA treatment and OGT knockdown altered proteins involved in critical pathways including DNA synthesome complex, glycolytic process, negative regulation of gene expression, cell cycle process, and negative regulation of protein phosphorylation. Specifically, OGT downregulated several ribosomal proteins, and OGT knockdown influenced proteins across the identified pathways. Taken together, these findings demonstrate that reducing OGT and protein O-GlcNAcylation may enhance the sensitivity of CRC cells to OXA, and the altered pathways may offer new insights into potential mechanisms for overcoming CRC chemoresistance.

There is increasing evidence of the health effects of germinated colored rice (GCR). Metastasis, namely the spread of cancer cells to distant sites in the body, is a serious problem for cancer therapy. This study aimed to explore the anti-metastatic potential of GCR extract in HT29 colorectal cancer cells through cell migration inhibition, and to identify target proteins by using proteomic approach. GCR extract at 4 mg/mL caused 87 % reduction in cell migration. Two-dimensional gel proteomic analysis revealed beta-actin downregulation in GCR-treated cells. GCR also affected gene expression of actin dynamic regulatory proteins including RHOA, RAC1, and CDC42 genes. Gamma-aminobutyric acid (GABA), a bioactive compound present in GCR extract, could decrease cell migration and downregulated beta-actin in the cells, but did not alter expression level of RHOA, RAC1, and CDC42 genes. This study reveals a potential of GCR to be developed as a functional food for assisting the cancer treatment.

The Para rubber tree (Hevea brasiliensis) is one of the most economically important latex-producing plants in the industrial sectors worldwide. The RRIT 251 clone is well recognized for its high latex yield and tolerance to diverse environmental conditions. Although considerable efforts have been made to enhance the latex production of RRIT 251, a comprehensive analysis of the functional and structural properties of its proteins remains limited. In this study, an in-depth proteomic analysis was conducted on C-serum (CS) and rubber particles (RPs) to gain insights into the molecular mechanisms and biological processes involved in latex production in the RRIT 251 clone. A total of 3028 proteins were identified in CS and 1854 proteins in RPs. Based on the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), many of proteins were preliminary classified into three groups: posttranslational modification, protein turnover and chaperones. A comprehensive analysis of protein domain family (Pfam) revealed that the protein kinase and small GTP-binding domains were most frequently detected in CS and RP fractions. In addition, KEGG and Plant Reactome pathway enrichment analyses of CS and RP fractions showed significant association with the Metabolism category. Collectively, these results suggest that proteins from the CS and RP fractions of latex play essential roles in cellular and metabolic processes. These findings not only deepen our understanding of the RRIT 251 latex proteome but also provide further insights into the biological processes underlying latex production and plant defense.

Exposure to pesticides has been considered as a risk factor for developing neurodegenerative diseases. The increasing use of fipronil, a phenylpyrazole insecticide, poses a risk to human health. This study aims to use toxicoproteomics for exploring neurodegenerative mechanism of fipronil in SH-SY5Y human neuroblastoma cells. In this study, fipronil at sub-cytotoxic and cytotoxic concentrations (43 and 78 μM) caused increases in superoxide level from 3 to 48 h after treatment, while intracellular glutathione level was decreased at 48 h. Neurite outgrowth of the cells was impaired by fipronil at both concentrations, while significant increase of cell death via apoptosis and necrosis modes were observed with fipronil at cytotoxic concentration. Pretreatment with antioxidant N-acetylcysteine (NAC) effectively relieved impairment of neurite outgrowth and induction of cell death by fipronil. Proteomic analysis showed that expression of proteins involving endoplasmic reticulum (ER) stress and unfolded protein responses were predominantly affected by fipronil. Immunoblotting confirmed the increased expression of ER stress markers, GRP78/BiP (78 kDa glucose-regulated protein/Binding immunoglobulin protein) and PDI (protein disulfide isomerase), in fipronil-treated cells. Improved understanding of the neurotoxic mechanism of fipronil may help in developing a strategy for reducing risk of neurodegenerative development from intense and prolonged use of fipronil.

Colorectal cancer (CRC) is a major global health issue due to its aggressiveness and high mortality rates. While curcumin, the bioactive compound from turmeric (Curcuma longa L.), exhibits anticancer properties, its low water solubility limits therapeutic potential. The present study developed a novel effervescent curcumin-ascorbic acid-polysaccharide-β-cyclodextrin (CUR-A-Poly-β-CD) inclusion complex to enhance solubility and evaluated its anticancer potential in CRC cell lines. The complex exhibited a zeta potential of -22.9 ± 1.1 mV, a particle size of 496.8 ± 1.9 nm, a solubility of 13.6 mg/L, and a pH of 4.5. CUR-A-Poly-β-CD was non-toxic to normal colon cells (CCD-841 CoN) but significantly reduced the viability of CRC cell lines HT-29, SW-48, and SW-480, with SW-480 cells being the most responsive. CUR-A-Poly-β-CD induced apoptosis and inhibited proliferation, clonogenicity, and migration of SW-480 cells. Proteomic analysis identified 105 altered proteins in SW-480 cells treated with CUR-A-Poly-β-CD, primarily involved in cell growth and ribosome biogenesis pathways. Western blot analysis confirmed increased levels of ribosomal proteins RPL13a and RPS3. In conclusion, the CUR-A-Poly-β-CD inclusion complex demonstrates anticancer effects against CRC by modulating ribosome biogenesis. These findings suggest its potential as a therapeutic approach to improve the effectiveness of CRC treatments.

SummarySplenectomised β‐thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra‐cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non‐splenectomised β‐thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)‐bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15–20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.

Winged bean (Psophocarpus tetragonolobus (L.) DC.) is a tropical legume crop found in Southeast Asian countries including Thailand. The mature seeds have high protein content and have been targeted as a protein source, and germination has been used to increase the value of nutrition. We used nanoLC/timsTOF Pro 2 combined with PEAKS® studio Xpro software to explore the proteomes of mature seeds and sprouts of purple winged bean. This study is the first report on the allergenic proteins in mature seeds and sprouts of purple winged bean. In silico analysis using Allergen Nomenclature search and the Allermatchtm program, 37 allergens were found. Conglutin beta 5 and late embryogenesis abundant protein (3 and D-29) were identified only in mature seeds, while 4 types of major pollen allergen Bet V, profilin and isoflavone reductase, were identified only in sprouts. Examples of allergens in both samples include basic 7S globulin, beta-conglycinin alpha/beta subunit (1,2), convicilin, defensin-like protein, 7 types of non-specific lipid-transfer protein, P24 oleosin isoform B. Since food allergy and airway allergy are found in sprouts, we used shotgun proteomics and in silico analysis for detecting the profiles of allergens. With regards to food safety, purple winged beans may also induce cross-reactivity in patients who are allergic to peanuts or other legumes. Therefore, identifying the proteomes and potential allergens in mature seeds and sprouts of purple winged beans could assist researchers in studying plant-based food safety.

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC-MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.

We recently reported that arsenic disrupted neuronal insulin signaling. Here, we further investigated the effect of arsenic on insulin receptor substrate (IRS) proteins, which are crucial downstream signaling molecules of insulin in differentiated human neuroblastoma SH-SY5Y cells. We also found that prolonged arsenic treatment accelerated the migration of IRS1 and IRS2 on SDS-PAGE. Treatment with phosphatases abolished the arsenic-induced increased mobility of IRS, suggesting that the electrophoretic mobility shift of IRS on SDS-PAGE by arsenic was phosphorylation-dependent. By using label-free mass spectrometry, the phosphorylation sites of IRS1 were found to be S24, S345, S636, T774, S1057, S1058, and S1070, while those of IRS2 were at S645, Y653, T657, S665, S667, S669, S672, S915, and S1203, which were at least 2-fold lower than found in the control. These findings indicated a global hypophosphorylation of IRS proteins after prolonged arsenic treatment. In addition, four novel phosphorylation sites were identified on IRS1 (T774, S1057, S1058, and S1070), with another two on IRS2 (S665 and S667). As basal IRS phosphorylation plays an important role in insulin signaling, the reduction of IRS phosphorylation on multiple residues may underlie arsenic-impaired insulin signaling in neurons.

Fish is an important food source but can contain allergens which cause mild to life-threatening symptoms. The aim of this study was to compare the identified proteins in fresh and powdered fish in skipjack tuna and Nile tilapia using proteomic approach. The biological functions of the proteins were identified using the UniProt database. Potential allergens were identified based on the sequence similarity to known allergens using the Allermatch program. The major functions of the identified proteins in both the fresh and powder forms of skipjack tuna and Nile tilapia were related to metabolic processes and the cytoskeleton. Eleven proteins in skipjack tuna and seven proteins in Nile tilapia, in fresh and powdered forms, were identified as novel potential allergens using the Allermatch program. Fructose-bisphosphate aldolase, enolase, parvalbumin, glyceraldehyde-3-phosphate dehydrogenase, and tropomyosin were found in abundance as potential allergens in both fishes. For both fishes, more isoforms of tropomyosin were found in powered fish than in fresh fish. Alpha- and beta-enolase, glyceraldehyde-3-phosphate dehydrogenase, alpha-, beta- and 2-parvalbumin, and tropomyosin alpha-1 chain could be developed as peptide markers for fish adulteration in both skipjack tuna and Nile tilapia. Interestingly, the peptides from eight potential allergens in fish were most similar to peptides from chicken powder and egg yolk powder. Here, proteomic analysis was used to study protein expression in skipjack tuna and Nile tilapia (both fresh and powdered). In addition, use of the untargeted proteomics approach could aid in the discovery of potential allergens and lead to the design of peptide markers to enhance food safety.

A shotgun proteomic and in-silico analysis was performed to identify and characterize the proteomes and potential allergens in both processed and non-processed forms of krill (Acetes spp.) and whiteleg shrimp (Litopenaeus vannamei). The samples were analyzed using LC-MS/MS, and the identified proteins were analyzed for protein-protein interactions and Gene Ontology. Potential allergens were analyzed by Allermatch and predicted IgE epitope by AlgPred. The results revealed that processed krill and whiteleg shrimp had a higher number of identified proteins and potential allergens compared to their non-processed forms. The major functions of identified proteins were related to organelle organization, intracellular and cytoskeletal protein binding. Eleven potential allergens were identified in all samples, indicating common allergens in both species. In-silico analysis of potential allergens of krill and whiteleg shrimp showed IgE epitopes on alpha-actinin sarcomeric-like isoform X1, tropomyosin fast isoform and tropomyosin isoform X19. The peptide IQLLEEDLER in tropomyosin fast isoform was identified as a potential allergen marker in fresh, dried and powdered forms of krill and whiteleg shrimp. These findings provide insight into the food safety, clinical and diagnostic aspects of krill and shrimp, particularly regarding its potential allergenicity and its allergen biomarkers.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Colorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Although commercial biomarkers of CRC are currently available, they are still lacking in terms of sensitivity and specificity; thus, searching for reliable blood-based biomarkers are important for the primary screening of CRC. Plasma samples of patients with non-metastatic (NM) and metastatic (M) CRC and healthy controls were fractionated using MARS-14 immunoaffinity chromatography. The flow-through and elute fractions representing low- and high-abundant proteins, respectively, were analyzed by label-free quantitative proteomics mass spectrometry. The functional analysis of the proteins with greater than 1.5-fold differential expression level between the CRC and the healthy control groups were analyzed for their biological processes and molecular functions. In addition, the levels of plasma proteins showing large alterations in CRC patients were confirmed by immunoblotting using two independent cohorts. Moreover, receiver operating characteristic (ROC) curve analysis was performed for individual and combinations of biomarker candidates so as to evaluate the diagnostic performance of biomarker candidates. From 163 refined identifications, five proteins were up-regulated and two proteins were down-regulated in NM-CRC while eight proteins were up-regulated and three proteins were down-regulated in M-CRC, respectively. Altered plasma proteins in NM-CRC were mainly involved in complement activation, while those in M-CRC were clustered in acute-phase response, complement activation, and inflammatory response. Results from the study- and validation-cohorts indicate that the levels of leucine-rich alpha-2-glycoprotein-1(LRG), complement component C9 (C9), alpha-1-acid glycoprotein 1 (AGP1), and alpha-1-antitrypsin (A1AT) were statistically increased, while fibronectin (FN) level was statistically decreased in CRC patients compared to healthy controls, with most alterations found in a metastatic stage-dependent manner. ROC analysis revealed that FN exhibited the best diagnostic performance to discriminate CRC patients and healthy controls while AGP1 showed the best discrimination between the disease stages in both cohorts. The combined biomarker candidates, FN + A1AT + AGP1, exhibited perfect discriminatory power to discriminate between the CRC population and healthy controls whereas LRG + A1AT + AGP1 was likely to be the best panel to discriminate the metastatic stages in both cohorts. This study identified and quantified distinct plasma proteome profiles of CRC patients. Selected CRC biomarker candidates including FN, LRG, C9, A1AT, and AGP1 may be further applied for screening larger cohorts including disease groups from other types of cancer or other diseases.

Supplementary Table S4: The expression levels of six proteins in individual plasma samples in the study cohort determined by westernblotting

NieR is a TetR family transcriptional repressor previously shown to regulate the NaOCl-inducible efflux pump NieAB in Agrobacterium tumefaciens. NieR is an ortholog of Escherichia coli NemR that specifically senses hypochlorite through the redox switch of a reversible sulfenamide bond between C106 and K175. The amino acid sequence of NieR contains only one cysteine. NieR has C104 and R166, which correspond to C106 and K175 of NemR, respectively. The aim of this study was to investigate the redox-sensing mechanism of NieR under NaOCl stress. C104 and R166 were subjected to mutagenesis to determine their roles. Although the substitution of R166 by alanine slightly reduced its DNA-binding activity, NieR retained its repressor function. By contrast, the DNA-binding and repression activities of NieR were completely lost when C104 was replaced by alanine. C104 substitution with serine only partially impaired the repressor function. Mass spectrometry analysis revealed an intermolecular disulfide bond between the C104 residues of NieR monomers. This study demonstrates the engagement of C104 in the mechanism of NaOCl sensing. C104 oxidation induced the formation of a disulfide-linked dimer that was likely to alter conformation, thus abolishing the DNA-binding ability of NieR and derepressing the target genes.

Nitrile Oxide−Dehydroalanine Cycloaddition is a bioconjugation strategy between dehydroalanine-containing proteins and nitrile oxides, remarkably, in fully aqueous-buffered systems without an additional catalyst. This novel method of chemical protein modification forms an isoxazoline ring enhancing the diversity of protein function and structure. The 1,3-dipolar cycloaddition reaction adds a fluorescent label to the protein for applications like live-cell imaging and apoptosis detection. More information can be found in the Research Article by A. Phanumartwiwath et al.