Search Results (35)

Background: Rabies is among the oldest known zoonotic viral diseases and is caused by members of the Lyssavirus genus. The prototype species, Lyssavirus rabies, effectively evades the host immune response, allowing the infection to progress unnoticed until the onset of clinical signs. At this stage, the disease is irreversible and invariably fatal, with definitive diagnosis possible only post-mortem. Given the advances in modern proteomics, this study aimed to identify potential protein biomarkers for antemortem diagnosis of rabies in dogs, which are the principal reservoir hosts of the rabies virus. Methods: Two hundred and thirty-one samples (brain tissues (BT), cerebrospinal fluids (CSF), and serum (SR) samples) were collected from apparently healthy dogs brought for slaughter for human consumption in South-East and North-Central Nigeria. All the BT were subjected to a direct fluorescent antibody test to confirm the presence of lyssavirus antigen, and 8.7% (n = 20) were positive. Protein extraction, quantification, reduction, and alkylation were followed by on-bead (HILIC) cleanup and tryptic digestion. The resulting peptides from each sample were injected into the Evosep One LC system, coupled to the timsTOF HT MS, using the standard dia-PASEF short gradient data acquisition method. Data was processed using Spectronaut^TM (v19). An unpaired t-test was performed to compare identified protein groups (proteins and their isoforms) between the rabies-infected and uninfected BT, CSF, and SR samples. Results: The study yielded 54 significantly differentially abundant proteins for the BT group, 299 for the CSF group, and 280 for the SR group. Forty-five overlapping differentially abundant proteins were identified between CSF and SR, one between BT and CSF, and two between BT and SR; none were found that overlapped all three groups. Within the BT group, 33 proteins showed increased abundance, while 21 showed decreased abundance in the rabies-positive samples. In the CSF group, 159 proteins had increased abundance and 140 had decreased abundance in the rabies-positive samples. For the SR group, 215 proteins showed increased abundance, and 65 showed decreased abundance in the rabies-positive samples. Functional enrichment analysis revealed that pathways associated with CSF, spinocerebellar ataxia, and neurodegeneration were among the significant findings. Conclusion: This study identified canonical proteins in CSF and SR that serve as candidate biomarkers for rabies infection, offering insights into neuronal dysfunction and potential tools for early diagnosis. Full article

Background/Objectives: Ergot alkaloids are mycotoxins produced mainly by fungi of the genus Claviceps, infecting a wide variety of plants, especially cereals. These toxins usually manifest as black, hardened sclerotia (ergots), though they may also be invisible when dispersed in grain. They pose a significant risk to animals and humans when present in contaminated cereals. They can cause ergotism, with vasoconstriction, ischemia, hallucinations, and in severe cases gangrene. This study was carried out in response to the European legislative actions which determine the permissible levels of ergot alkaloids in cereals. Historically, consumers manually removed visible sclerotia from grain, and farmers applied fertilizers or timed harvests to specific periods to mitigate contamination. However, these traditional methods have proven insufficient. We therefore explored advanced techniques for detecting and quantifying ergot-contaminated cereals, as well as methods for reducing ergot alkaloid concentrations. Methods: Searches were conducted in scientific databases including Google Scholar, PubMed, and Scopus to identify research articles, reviews, and experimental studies published mainly between 2012 and August 2025, including accepted or in-press manuscripts, with special attention to works from 2021 onward to capture the most recent advancements. Results/Conclusions: Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) is the reference method for confirmatory, epimer-aware quantification of ergot alkaloids, and is already standardized. Recent QuEChERS-UHPLC-MS/MS workflows in cereal matrices, including oat-based products, routinely achieve limits of quantification of about 0.5–1.0 µg/kg with single-run analysis times of about 5–15 min. Rapid screening options complement, rather than replace, confirmatory mass spectrometry: magnetic bead-based immunoassays that use magnetic separation and a smartphone-linked potentiostat provide sub-hour turnaround and field portability for trained quality-assurance staff, although external validation and calibration traceable to LC-MS/MS remain prerequisites for routine use. In practice, operators are adopting tiered, orthogonal workflows (e.g., immunoassay or electronic-nose triage at intake followed by DNA-based checks on grain washings and LC–MS/MS confirmation, or hydrazinolysis “sum parameter” screening followed by targeted MS speciation). Such combinations reduce turnaround time while preserving analytical rigor. Biotechnology also offers potential solutions for reducing ergot alkaloid concentrations at the source. Finally, to enhance consumer safety, artificial intelligence and blockchain-based food traceability appear highly effective. These systems can connect all stakeholders from producers to consumers, allowing for real-time updates on food safety and rapid responses to contamination issues. This review primarily synthesizes advances in analytical detection of ergot alkaloids, while mitigation strategies and supply chain traceability are covered concisely as supporting context for decision making. 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

As a type II ribosome-inactivating protein (RIP-II) toxin, Ricin has garnered widespread recognition due to its inherent qualities as an easily prepared and highly stable substance, posing serious implications as a potential chemical and biological terrorist threat. For the detection of ricin, traditional immunoassay technologies, including methods like peptide cleavage combined with liquid chromatography mass spectrometry (LC-MS) or the more commonly used enzyme-linked immunosorbent assay (ELISA), have offered reliable results. However, these techniques are unfortunately limited by the requirement of a complex sample pretreatment process, which can be time-consuming and labor-intensive. In an effort to overcome these limitations, a highly sensitive and selective method was introduced via metal element labeling combined with inductively coupled plasma mass spectrometry (ICP-MS) in this research. The method centered on designing and synthesizing a europium-labeled compound (DOTA-NHS-Eu) that specifically targets the amino groups (-NH₂) on ricin. The compound, coupled with the application of specific magnetic beads, achieved the specific enrichment and subsequent quantitative detection of ricin by ICP-MS, which is based on the amount of europium element present. The established method demonstrated high specificity for ricin recognition, with a signal response to bovine serum protein that was found to be less than 10% of that for ricin. Furthermore, the calibration curve created for the method (y = 81.543x + 674.02 (R² > 0.99)) for quantifying ricin in a concentration range of 1.0–100 μg/mL demonstrated good linearity. The method was further evidenced by the limit of detection and quantitation results of 0.1 and 1.89 μg/mL, respectively. Collectively, these findings suggested that the research has offered a highly sensitive and selective method for ricin detection, which was not only easy to operate but also provided efficient results. The scheme showed great potential for the verification of chemical weapons and the destruction of toxic chemicals, therefore representing a significant advancement in the field of biomolecular detection and analysis. Full article

Background/Objectives: Ruptured abdominal aortic aneurysm (RAAA) remains a leading cause of vascular death, with mortality rates approaching 90%. Biomarkers capable of identifying the most at-risk population are urgently needed in the clinic. We aimed to identify potential alterations in the urine proteome that can enable non-invasive detection of abdominal aortic aneurysms (AAA) at high risk of rupture. Methods: We used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MIB/MS) to examine potential biomarkers in urine samples. Quantitative proteomic profiling was conducted using iTRAQ labeling and LC-TEMPO MALDI-TOF/TOF analysis, revealing several dysregulated proteins in the urinary proteome between the two groups. MS and MS/MS data were generated using MALDI TOF/TOF instruments (models 5800 or 4800; AB SCIEX). MS/MS spectra were processed with ProteinPilot™ software version 3.0 (AB SCIEX) and matched against the UniProt/Swiss-Prot database for identification of proteins with an Unused ProtScore >1.3. Statistical tests were performed using R/Bioconductor software and bioinformatics analysis using open-source software. Results: We quantitatively measured activity over 130 kinases from various kinase families using MIB/MS with a threshold of 1.5-fold change in expression. Statistical analysis assigned significance to EPHB6, AXL, EPHB4, DDR1, EPHA2 and EPHB3. All were tyrosine kinases, and the Ephrin receptor type was dominant. The reduced expression of specific kinases identified by MIB/MS analysis was validated by Western blot. Conclusions: This pilot study presents a promising breakthrough in the diagnosis and surveillance of AAA. We identified six dysregulated tyrosine kinases in the urine proteome of patients with RAAAs, suggesting their potential as urinary biomarkers for early detection of AAA at high risk of rupture. However, these preliminary findings require confirmation in larger, prospective cohorts to validate their diagnostic utility and generalizability. Full article

An eco-friendly extraction process of polyphenols from conventional dried rosemary tissues and post-distillation waste residues was applied using β-cyclodextrin as a co-solvent. The aqueous extracts were characterized by measuring the total phenolic content, and their phenolic compounds were identified and quantified by LC-MS. Sodium alginate solutions (2% w/w) with/without incorporation of rosemary aqueous extracts were prepared and used for the preparation of O/W emulsions containing 20% rapeseed oil and an 80% water phase. Hydrogel beads were then stored at 20 °C for 28 days. The quality of encapsulated oil during storage was evaluated by measurements of the peroxide value, p-anisidine value, free fatty acids, total oxidation value, and fatty acid composition, whilst the aqueous phase of the beads was analyzed for its total extractable phenolic content (TEPC). The experimental findings indicate that the incorporation of aqueous extracts from post-distillation rosemary residues in emulsion-filled hydrogel beads resulted in the lowest level of oxidation products in the encapsulated rapeseed oil (PV = 10.61 ± 0.02 meq/Kg oil, p-AnV = 4.41 ± 0.09, and FFA = 0.14 ± 0.00, expressed as % oleic acid content), indicating an acceptable oil quality until the end of the storage period. Full article

Lung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem–loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method’s performance in more complex biological samples, including A549 cells’ total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells’ total RNA. Full article

The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction. Full article

Background: Tacrolimus (TAC) and mycophenolic acid (MPA) are commonly used immunosuppressive therapies after renal transplant. Our objective was to quantify TAC and MPA concentrations in peripheral blood mononuclear cells (PBMCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) and to evaluate and validate the performance of the methodology. A prospective follow-up cohort study was conducted to determine whether intracellular concentrations were associated with adverse outcomes in renal transplants. Methods: PBMCs were prepared using the Ficoll separation technique and purified with erythrocyte lysis. The cells were counted using Sysmex XN-3100 and then packaged and frozen according to a 50 µL volume containing 1.0 × 10⁶ cells. TAC and MPA were extracted using MagnaBeads and quantified using an LC-MS/MS platform. The chromatography was run on a reversed-phase Waters Acquity UPLC BEH C18 column (1.7 µm, 50 mm × 2.1 mm) for gradient elution separation with a total run time of 4.5 min and a flow rate of 0.3 mL/min. Mobile phases A and B were water and methanol, respectively, each containing 2 mM ammonium acetate and 0.1% formic acid. Renal transplant recipients receiving TAC and MPA in combination were selected for clinical validation and divided into two groups: a stable group and an adverse outcome group. The concentrations were dynamically monitored at 5, 7, 14, and 21 days (D5, D7, D14, and D21) and 1, 2, 3, and 6 months (M1, M2, M3, and M6) after operation. Results: Method performance validation was performed according to Food and Drug Administration guidelines, showing high specificity and sensitivity. The TAC and MPA calibration curves were linear (r² = 0.9988 and r² = 0.9990, respectively). Both intra-day and inter-day imprecision and inaccuracy were less than 15%. Matrix effects and recoveries were satisfactory. The TAC and MPA concentrations in 304 “real” PBMC samples from 47 renal transplant recipients were within the calibration curve range (0.12 to 16.40 ng/mL and 0.20 to 4.72 ng/mL, respectively). There was a weak correlation between PBMC-C_0TAC and WB-C_0TAC (p < 0.05), but no correlation was found for MPA. The level of immunosuppressive intra-patient variation (IPV) was higher in PBMC at 77.47% (55.06, 97.76%) than in WB at 34.61% (21.90, 49.85%). During the dynamic change in C_0TAC, PBMC-C_0TAC was in a fluctuating state, and no stable period was found. PBMC-C_0TAC did not show a significant difference between the stable and adverse outcome group, but the level of the adverse outcome group was generally higher than that of the stable group. Conclusions: Compared with conventional therapeutic drug monitoring, the proposed rapid and sensitive method can provide more clinically reliable information on drug concentration at an active site, which has the potential to be applied to the clinical monitoring of intracellular immunosuppressive concentration in organ transplantation. However, the application of PBMC-C_0TAC in adverse outcomes of renal transplant should be studied further. Full article

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp’s conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp’s solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins. Full article

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab’)₂) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab’)₂ was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R² > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858–0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods. Full article

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1β were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation. Full article

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3′,4′:6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38. Full article

Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC–MS/MS analysis, and data analysis. LC–MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation. Full article

Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen–antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody–drug conjugate targeting of these specific proteins may be promising for clinical applications. Full article