Search Results (57)

Recent advancements in neuroproteomics have enabled detailed analysis of protein expression in the human brain, yet resolving synaptic dysfunction—a central feature of many neurological and psychiatric disorders—requires careful methodological consideration. Leveraging the high sensitivity of modern liquid chromatography-tandem mass spectrometry (LC-MS/MS), we evaluated the utility of whole-tissue lysates versus enriched synaptosome preparations for detecting synaptic protein signatures. First, we optimized and standardized a sample preparation protocol for frozen human gray matter (GM) by refining the suspension trapping (sTRAP) digestion method using thin human tissue sections. We accomplished low technical variation by minimizing sample handling and achieved a highly reproducible sample preparation workflow by rigorously applying standardization and randomization across dissection, processing, and LC-MS/MS runs. Second, comparative LC-MS/MS analysis showed that while whole-tissue lysates provide a high-throughput survey of the synaptic proteome, synaptosome isolation is required to investigate synapse-specific proteins to detect alterations at the terminal that are obscured in the soma. Because these methods offer distinct but synergistic levels of information, we recommend a tiered neuroproteomics strategy. This approach utilizes whole-tissue lysates for broad disease-associated screening and consistent quantification in large cohorts, followed by targeted synaptosome proteomics to provide a unique window of insight into synaptic composition and stability. This integrated workflow respects the biological necessity of spatial resolution while maintaining the reproducibility required for robust human brain proteomics. Furthermore, initial tissue-level analysis provides the necessary context to correctly interpret synaptosome data in cases of global synapse loss or gain. Full article

The ‘Pingguoli’ pear (Pyrus pyrifolia cv. Pingguoli) has a cultivation history spanning nearly one hundred years. Bud mutation selection is an important breeding method for the ‘Pingguoli’ pear. In this study, high-throughput sequencing technology (RNA-Seq) and non-targeted metabolomics (LC-MS/MS) were used to analyze the large-fruited bud mutation line (LFS) and normal type (NTF) of the ‘Pingguoli’ pear during the cell division (G1), rapid growth (G2), and mature stages (G3) of the fruit. The results showed that LFS exhibited a 46.32% increase in average single fruit weight (383.01 ± 54.72 g vs. 261.76 ± 10.79 g, p < 0.01) and a 19.10% decrease in soluble solids content (12.70 ± 0.94% vs. 15.40 ± 2.06%, p < 0.05) compared to NTF. Compared with the NTF, the content of total phenols and total flavonoids and the activity of antioxidant enzymes in the LFS fruits were significantly higher, while the contents of soluble sugar, reducing sugar, and soluble protein were significantly lower. Transcriptome analysis revealed that key metabolic pathways—including pentose and glucuronate interconversions, starch and sucrose metabolism, and cutin, suberine, and wax biosynthesis—were significantly enriched between NTF and LFS. These pathways may contain the specific differentially expressed genes (e.g., those involved in sugar metabolism and wax biosynthesis) identified as potential regulators of fruit size, appearance, and nutritional quality in the LFS. LC-MS/MS analysis identified key differentially accumulated metabolites, including L-arginine, caffeic acid, L-cysteine, pyridoxamine 5′-phosphate, adenosine-5′-phosphosulfate, neopentolactone D, chlorogenic acid, and gluconic acid, which are directly associated with the nutritional and antioxidant differences between LFS and NTF. The genes most related to metabolites in the three different developmental periods of the LFS and NTF were identified through combined analysis. These results provide insights for further research on bud mutation breeding and the quality formation mechanism of ‘Pingguoli’ pears. Full article

The global edible oil market is consistently at risk of economically motivated adulteration, underscoring the necessity of robust analytical methods essential for authentication. Among various phytochemicals, phytosterols have emerged as powerful diagnostic markers and compositional indicators for verifying the botanical origin, purity, and quality of edible oils. This review summarizes recent advancements in phytosterol analysis, highlighting its application in detecting adulteration in high-value oils such as olive oil, tea seed oil, and sesame oil. We discuss the approaches of multiple chromatographic and mass spectrometry techniques (GC-MS, LC-MS) with chemometric analysis of novel markers like fatty acyl sterol esters and sterol degradation products. Furthermore, we discuss significant challenges, including the need for comprehensive databases, the identification of complex sterol compositional profiles, and the limitations of current standardized methods. The advancement of phytosterol-based authentication increasingly depends on the development of rapid, high-throughput, and non-targeted sterol profiling approaches, supported by artificial intelligence and bioinformatics, to ensure vegetable oil authenticity and safeguard market integrity. Full article

The Lushan Russet potato, cultivated in Lushan Mountain (China), is renowned for its unique flavor, which deteriorates when cultivated at low altitudes. To unravel its flavor determinants, we compared high/low-altitude-cultivated Lushan Russet potato (LsM/LS) and reference Zhongshu5 (Zs) via nutritional, metabolomic, and endophytic microbiota analyses. LsM/LS had higher dry matter, potassium, and other flavor-related components than Zs. Non-targeted LC-MS metabolomics identified 461 metabolites. Pairwise comparisons revealed 263 significant differential metabolites (SDMs) between LsM and Zs (205 more abundant in LsM), 240 between LS and Zs, and 237 between LsM and LS. KEGG enrichment showed that SDMs were mainly involved in metabolic pathways. High-throughput sequencing of endophytic microbiota showed clear beta diversity separation, which correlated with metabolomic changes. These results indicate that Lushan Russet potato’s unique flavor is jointly determined by nutrient/metabolite accumulation and endophytic microbiome diversity, providing a basis for optimizing its quality and mitigating flavor deterioration in low-altitude cultivation. Full article

Background/Objectives: Different oxylipin subtypes have unique biological properties, requiring effective analytical protocols. However, establishing a complete pathway detection protocol for comprehensive oxylipin analysis is challenging. This study aimed to evaluate the adaptability and specificity of oxylipin subtypes under different extraction schemes and to develop a robust analytical platform for clinical biomarker investigation. Methods: We revealed the adaptability and specificity of oxylipin subtypes based on different single-step extraction schemes. A high-throughput quantitative automated solid-phase extraction coupled with a liquid chromatography–tandem mass spectrometry (aSPE–LC–MS/MS) analytical platform was established for a broad panel of complex oxylipins. The method was applied to serum samples of patients with coronary heart disease (CHD). Results: Our results verified that oxo-oxylipins, resolvin, and eicosanoids showed the best extraction efficiency under SPE protocol. Most hydroxy-oxylipins, dihydroxy-oxylipins, and HOTrEs are suitable for methanol protocol, HDHA for acetonitrile protocol, and epoxy-oxylipins for the methyl tert-butyl ether protocol, while medium-chain HETE is suitable for ethyl acetate protocol. Importantly, a novel sensitive fast method with wide coverage by the aSPE–LC–MS/MS analytical platform with satisfying sensitivity, accuracy and precision, extraction efficiency, low matrix effect, and linear calibration curves was obtained. Furthermore, we have successfully applied this method and found that 5-HETE, 11-HETE, and 15-HETE can serve as integrated biomarkers for patients with CHD, with high diagnostic performance. Conclusions: The study provides the best protocol for the clinically targeted detection of oxylipins and provides an important means for studying biomarkers of diseases. Full article

Occupational exposure to soil-borne pesticides remains a critical safety and process-management challenge in industrial and agro-industrial settings. This work proposes a process-integrated analytical workflow that couples comparative instrumental identification of soil xenobiotics with an occupational risk assessment framework. We comparatively evaluate GC-MS (gas chromatography–mass spectrometry), HPLC (high performance liquid chromatography), FTIR (Fourier-Transform Infrared Spectroscopy), LC-MS/MS (Liquid Chromatography coupled with tandem Mass Spectrometry), and ICP-MS (Inductively Coupled Plasma Mass Spectrometry) against matrix complexity, sensitivity, cost, and throughput, and implement the Quick, easy, cheap, effective, rugged, safe (QuEChERS) method-based sample preparation followed by GC-MS and LC-MS/MS to demonstrate applicability on representative soil and food-chain samples. Complementary risk tools (toxicity–probability matrices, exposure pathway diagrams) and an integrated monitoring scheme that combines environmental data with biomonitoring are used to link concentrations to exposure potential and control priorities. In a soil case sample, low-level organochlorines were detected with total DDT at 0.010 mg/kg and total HCH at 0.003 mg/kg, illustrating how analytical outputs feed decision matrices for prioritizing interventions. Case analyses from agricultural and industrial contexts indicate that targeted substitution, optimized application, ventilation and dust control, PPE (personal protective equipment) adherence, and worker training can measurably reduce symptoms and biomarkers of exposure. Overall, a complementary, process-analytical approach—integrating sensitive multi-technique detection with exposure assessment and continuous monitoring—supports proactive risk management and aligns with process systems and monitoring themes. Recommendations include standardizing workflows, coupling routine environmental monitoring with biomonitoring where feasible, and embedding preventive policies and training into industrial management systems. Full article

Pharmaceuticals and personal care products (PPCPs) are recognized as emerging contaminants of concern, even at ultra-trace concentrations. However, the current detection systems are prohibitively expensive and typically rely on labor-intensive, lab-based workflows that lack automation in sample pretreatment. In this study, we developed a robotic and on-flow solid-phase extraction (ROF-SPE) system, fully integrated with online liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the on-site and real-time monitoring of 16 PPCPs in wastewater effluent. The system automates the entire pretreatment workflow—including sample collection, filtration, pH adjustment, solid-phase extraction, and injection—prior to seamless coupling with LC–MS/MS analysis. The optimized pretreatment parameters (pH 7 and 10, 12 mL wash volume, 9 mL elution volume) were selected for analytical efficiency and cost-effectiveness. Compared with conventional offline SPE methods (~370 min), the total analysis time was reduced to 80 min (78.4% reduction), and parallel automation significantly enhanced the throughput. The system was capable of quantifying target analytes at concentrations as low as 0.1 ng/L. Among the 16 PPCPs monitored at a municipal wastewater treatment plant in South Korea, only sulfamethazine and ranitidine were not detected. Compounds such as iopromide, caffeine, and paraxanthine were detected at high concentrations, and seasonal variation patterns were also observed This study demonstrates the feasibility of a fully automated and on-site SPE pretreatment system for ultra-trace environmental analysis and presents a practical solution for the real-time monitoring of contaminants in remote areas. Full article

Coffee adulteration is a growing concern in the food industry due to economic and quality implications. This study evaluates a rapid, non-targeted fingerprinting method based on flow injection analysis–mass spectrometry (FIA-MS) for detecting common coffee adulterants. A total of 119 samples were analyzed, including 43 coffee samples and 76 samples of common coffee adulterants (16 chicory, 10 barley, and 50 flour samples). FIA-MS combined with chemometric analysis allowed for the classification of pure and adulterated coffee samples with over 95% accuracy. Compared to LC-MS, the FIA-MS method showed a similar performance while offering significantly faster analysis and lower solvent consumption, making it a practical and sustainable option for high-throughput screening. For PLS regression studies, calibration and prediction errors were consistently below 0.91% and 11.7%, respectively. Furthermore, the methodology was compared with a non-targeted LC-MS approach, showing an excellent performance. 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

Background: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency. Methods: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach’s applicability in putative biomarker discovery. Results: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness. Conclusions: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential. Full article

Sitagliptin is an orally bioavailable selective DPP4 inhibitor that reduces blood glucose levels without significant increases in hypoglycemia. The aim of this study was to design and validate an innovative, rapid, and highly sensitive LC–MS/MS assay for the precise measurement of sitagliptin concentrations in human plasma. This analytical method, utilizing sitagliptin-d4 as the internal standard, is performed using only 100 μL of plasma and a liquid–liquid extraction procedure based on methyl tert-butyl ether (MTBE). Chromatographic separation is expertly achieved with a Kinetex^® C18 column under isocratic elution, employing a perfect 1:1 blend of 5 mM ammonium acetate (with 0.04% formic acid) and acetonitrile, and maintaining an efficient flow rate of 0.2 mL/min. Detection occurs in positive ionization mode through multiple reaction monitoring, precisely targeting transitions of m/z 408.2 → 193.0 for sitagliptin and 412.2 → 239.1 for the IS. The total runtime of this assay is under 2 min. Comprehensive validation in line with MFDS and FDA criteria demonstrates outstanding linearity (5–1000 ng/mL, r² > 0.998), alongside impressive levels of accuracy, precision, recovery and sample stability. Due to its minimal sample requirement and high-throughput capability, the validated approach is highly appropriate for pharmacokinetic and bioequivalence assessments involving sitagliptin. Full article

Mycotoxins are responsible for a multitude of diseases in both humans and animals, resulting in significant medical and economic burdens worldwide. Conventional detection methods, such as enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS), are highly effective, but they are generally confined to laboratory settings. Consequently, there is a growing demand for point-of-care testing (POCT) solutions that are rapid, sensitive, portable, and cost-effective. Lateral flow assays (LFAs) are a pivotal technology in POCT due to their simplicity, rapidity, and ease of use. This review synthesizes data from 78 peer-reviewed studies published between 2015 and 2024, evaluating advances in nanoparticle-based LFAs for detection of singular or multiplex mycotoxin types. Gold nanoparticles (AuNPs) remain the most widely used, due to their favorable optical and surface chemistry; however, significant progress has also been made with silver nanoparticles (AgNPs), magnetic nanoparticles, quantum dots (QDs), nanozymes, and hybrid nanostructures. The integration of multifunctional nanomaterials has enhanced assay sensitivity, specificity, and operational usability, with innovations including smartphone-based readers, signal amplification strategies, and supplementary technologies such as surface-enhanced Raman spectroscopy (SERS). While most singular LFAs achieved moderate sensitivity (0.001–1 ng/mL), only 6% reached ultra-sensitive detection (<0.001 ng/mL), and no significant improvement was evident over time (ρ = −0.162, p = 0.261). In contrast, multiplex assays demonstrated clear performance gains post-2022 (ρ = −0.357, p = 0.0008), largely driven by system-level optimization and advanced nanomaterials. Importantly, the type of sample matrix (e.g., cereals, dairy, feed) did not significantly influence the analytical sensitivity of singular or multiplex lateral LFAs (Kruskal–Wallis p > 0.05), confirming the matrix-independence of these optimized platforms. While analytical challenges remain for complex targets like fumonisins and deoxynivalenol (DON), ongoing innovations in signal amplification, biorecognition chemistry, and assay standardization are driving LFAs toward becoming reliable, ultra-sensitive, and field-deployable platforms for high-throughput mycotoxin screening in global food safety surveillance. Full article

Small molecule inhibitors that target the E3 ligase activity of MDM2-MDM4 have been explored to inhibit the oncogenic activity of MDM2-MDM4 complex. MMRi62 is a small molecule that was identified using an MDM2-MDM4 E3 ligase-based high throughput screen and a cell-death-based secondary screen. Our previous studies showed that MMRi62 promotes MDM4 degradation in cells and induces p53-independent apoptosis in cancer cells. However, MMRi62 activity in solid tumor cells such as melanoma cells, especially in BRAF inhibitor resistant melanoma cells, have not been explored. Although its promotion of MDM4 degradation is clear, the direct MMRi62 targets in cells are unknown. In this report, we show that MMRi62 is a much more potent p53-independent apoptosis inducer than conventional MDM2 inhibitors in melanoma cells. A brief structure-activity study led to development of SC-62-1 with improved activity. SC-62-1 potently inhibits and eliminates clonogenic growth of melanoma cells that acquired resistance to BRAF inhibitors. We developed a pair of active and inactive SC-62-1 probes and profiled the cellular targets of SC-62-1 using a chemical biology approach coupled with IonStar/nano-LC/MS analysis. We found that SC-62-1 covalently binds to more than 15 hundred proteins in cells. Pathways analysis showed that SC-62-1 significantly altered several pathways including carbon metabolism, RNA metabolism, amino acid metabolism, translation and cellular response to stress. This study provides mechanistic insights into the mechanisms of action for MMRi62-like quinolinols. This study also suggests multi-targeting compounds like SC-62-1 might be useful for overcoming resistance to BRAF inhibitors for improved melanoma treatment. Full article

Saffron is a high-cost spice due to the specific conditions for optimal growth and because of being harvested by hand. The massive income from commercializing saffron substituted with other plant parts or low-cost spices makes this spice the main target of fraudsters. Background: Different methods have been developed for detecting saffron adulteration. Most of them are time consuming and complex, and in some types of analysis, the whole untargeted dataset is combined with advanced chemometric tools to differentiate authentic from non-authentic saffron. The official method, combining UV–vis spectroscopy and LC to determine the colour strength and the crocin content, is unable to detect saffron adulterants (safflower, marigold, or turmeric) added at a level lower than 20% (w/w). As a result, innovative approaches based on rapid, high-throughput methods for the identification of adulterated saffron samples are urgently demanded to counteract food frauds. Methods: This paper describes, for the first time, the development of a method combining Direct Analysis in Real Time (DART) with the triple quadrupole MS EVOQ based on the detection of specific MS/MS transitions, promoting a rapid, robust and chromatography-free method capable of monitoring safflower and turmeric adulteration in saffron. Results: The method proved to reach low LODs, allowing the determination of tiny amounts of turmeric and safflower powder in saffron as low as 3% and 5%, respectively, speeding up the whole analytical workflow and enabling us to perform 20 analyses in 10 min. Finally, the greenness of the method was also assessed according to the 0.88 score achieved by submitting it to the greenness calculator AGREE. Conclusions: Given its speed, simplicity, and robustness, this method stands out as a strong candidate for routine implementation in testing laboratories as a rapid screening tool to detect saffron adulteration with safflower or turmeric. Full article

Background: Ischemic stroke (IS) is a severe condition with limited therapeutic options. Pyroptosis, a type of programmed cell death linked to inflammation, is closely associated with IS-related damage. Studies suggest inflammation aligns with the traditional Chinese medicine (TCM) concept of “fire-heat syndrome”. Huanglian Jiedu Decoction (HLJD), a TCM formula known for clearing heat and purging fire, has shown therapeutic effects on IS, potentially by regulating pyroptosis. Study design: Eight-week-old male mice were divided into six groups: sham operation, model, positive drug, and low-, medium-, and high-dose HLJD groups. After a week of adaptive feeding, mice received respective treatments for five days, followed by modeling on the sixth day, with samples collected 23 h post-perfusion. Analyses included multi-omics, physiology, histopathology, virtual drug screening, target affinity assessment, and molecular biology techniques to measure relevant indicators. Results: HLJD effectively mitigated IS-related damage, maintaining neurological function, reducing ischemic levels, protecting cellular morphology, inhibiting neuronal apoptosis, and preserving blood–brain barrier integrity. Bioinformatics of high-throughput omics data revealed significant activation of pyroptosis and related inflammatory pathways in IS. ScRNA-seq identified neutrophils, macrophages, and microglia as key pyroptotic cell types, suggesting potential therapeutic targets. Network pharmacology and molecular docking identified NLRP3 as a critical target, with 6819 ligand–receptor docking results. SPR molecular fishing, LC-MS, molecular dynamics, and affinity measurements identified small molecules with high affinity for NLRP3. Molecular biology techniques confirmed that HLJD regulates pyroptosis via the classical inflammasome signaling pathway and modulates the inflammatory microenvironment. Conclusions: Following IS, pyroptosis in myeloid cells triggers an inflammatory cascade, leading to neural damage. HLJD may inhibit NLRP3 activity, reducing pyroptosis and associated inflammation, and ultimately mitigating damage. Full article