Liquid Chromatography-tandem Mass Spectrometry Research Articles

A Novel Strategy for the Detection of Semicarbazide in Crustaceans by Modified QuEChERS Coupled with Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

Semicarbazide (SEM), a metabolite of nitrofurazone (NFZ), is widely used to detect the illegal application of NFZ in crustaceans. The conventional detection method involves chemical derivatization combined with reversed-phase liquid chromatography–tandem mass spectrometry (RPLC-MS/MS), which is both complex and time-consuming. To address this limitation, a more efficient approach was developed for SEM detection. This study introduces a modified QuEChERS pretreatment method coupled with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) for detecting SEM in crustaceans. The proposed method is simple, fast, and highly accurate, making it universally applicable for SEM detection in crustaceans. Additionally, the method was applied to investigate NFZ metabolism in Macrobrachium rosenbergii with a kinetic model. The findings suggested a plausible mechanism for the absorption of NFZ and its subsequent transfer from meat to the shell. In conclusion, this study provides a simple and rapid technique for SEM detection in crustaceans with immense application value.

A Novel LC-MS/MS Method for the Measurement of Elexacaftor, Tezacaftor and Ivacaftor in Plasma, Dried Plasma Spot (DPS) and Whole Blood in Volumetric Absorptive Microsampling (VAMS) Devices

Background: The combination of ivacaftor, tezacaftor and elexacaftor (ETI) is approved for patients with cystic fibrosis (CF) aged two years and older and at least one F508del mutation in the CFTR gene. Variability in ETI treatment response has been repeatedly reported, and its reasons are unclear and understudied. Objectives: We present a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid and simultaneous quantification of ETI in plasma, dried plasma spots (DPS), and whole blood volumetric absorptive microsampling (VAMS). Methods: The method utilizes a rapid extraction protocol with 200 μL methanol after the addition of deuterated internal standards. Chromatographic separation was achieved using a reversed-phase Hypersil Gold aQ column (Thermo Fisher Scientific). The method was validated according to ICH (International Council on Harmonisation) guidelines M10 for bioanalytical method validation, demonstrating linearity in the concentration range 0.020–12.000 µg/mL. It was also proved accurate and reproducible with no matrix effect. This method was applied to anonymized samples from patients undergoing ETI treatment: eight plasma and DPS and five VAMS samples were analyzed. Results: ETI concentrations measured in plasma and DPS were interchangeable, whereas ETI concentrations in VAMS were lower than in plasma, as expected for molecules with high plasma protein binding (99%). A correction factor based on the hematocrit value was used to calculate the equivalent plasma concentration from VAMS concentrations. Conclusions: This method is suitable for pharmacokinetic (PK) studies and could facilitate the centralization of samples to specialized laboratories, supporting multicenter studies.

Loss of Endothelial TRPC1 Induces Aortic Hypercontractility and Hypertension.

The increasing prevalence of obesity-related cardiovascular diseases demands a better understanding of the contribution of different cell types to vascular function for developing new treatment strategies. Previous studies have established a fundamental role of TRPC1 (transient receptor potential channel canonical family member 1) in blood vessels. However, little is known about its functional roles within different cell types. We generated endothelial-specific TRPC1-deficient and knockin mice and analyzed their changes in vascular function under physiological and pathologically obese state. Wire myography, Ca2+ image, blood pressure measurements, RNA-sequencing analysis, liquid chromatography-mass spectrometry, immunoblotting, ELISA, luciferase reporter assay, and morphometric assessments were performed to unravel phenotype and molecular changes in response to the absence or presence of endothelial TRPC1. Loss of endothelial TRPC1 reduced endothelial-dependent relaxation and exaggerated endothelial-dependent contraction in mouse aorta. As expected, loss of endothelial TRPC1 amplified blood pressure and decreased acetylcholine-induced intracellular Ca2+ concentration rise in the aorta. In endothelial-specific TRPC1-deficient mouse arteries, the mRNA profile identified upregulation of c-Fos. Blockade of c-Fos rescued the impaired vasomotor tone in the aorta of mice deficient in endothelial TRPC1. Endothelial TRPC1-regulated nitric oxide/endothelin-1 production is involved in vascular c-Fos expression. Moreover, knockin of endothelial TRPC1 ameliorated enhanced endothelial-dependent contraction and hypertension in obese mice which is related to alleviated endothelial endothelin-1/c-Fos production and smooth muscle contraction. Our results identify endothelial TRPC1 as a previously unclear regulator of vascular changes and blood pressure in both physiological and pathologically obese state, and it is associated with nitric oxide/endothelin-1/c-Fos signaling.

In vitro anticancer effects of frankincense and its nanoemulsions for enhanced cancer cell targeting

IntroductionFrankincense has demonstrated promising in vitro anticancer activity. However, its conventional delivery methods face significant challenges due to limited oral bioavailability. To address these limitations, this study focuses on developing optimized nanoemulsions (NEs) of Frankincense oil (FO) to enhance its therapeutic efficacy.MethodsFrankincense resins were extracted and characterized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), identifying key metabolites including isopinocarveol, α-thujene, p-cymene, carvone, germacrene A, and various methyl esters. FO-based nanoemulsions (FO-NEs) were prepared and optimized using a 3-factor, 3-level Box-Behnken Design (BBD), with 10% FO (v/v), 40% surfactant (cremophor EL), and co-surfactant (Transcutol P). The optimized FO-NEs were evaluated for particle size, polydispersity index (PDI), zeta potential, and morphology using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cytotoxicity, wound healing, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) assays were performed against breast cancer (MDA-MB-231, MDA-MB-231-TR) and lung cancer (A549, A549-TR, H1299) cell lines.ResultsThe optimized FO-NEs exhibited an average particle size of 65.1 ± 4.21 nm, a PDI of 0.258 ± 0.04, and a zeta potential of −22.3 ± 1.2 mV. SEM and AFM confirmed the spherical morphology of the FO-NEs. In vitro cytotoxicity studies revealed enhanced anticancer activity of FO-NEs (IC50 = 13.2 μg/mL) compared to free FO (IC50 = 22.5 μg/mL) against resistant breast cancer MDA-MB-231-TR cells. FO-NEs significantly improved cancer cell internalization, disrupted mitochondrial membrane potential, and increased ROS generation, leading to enhanced cytotoxic effects.DiscussionThe results demonstrate that nanoemulsion-based delivery significantly enhances the bioactivity and cellular uptake of frankincense oil compared to its free form. FO-NEs exhibit potent anticancer activity, particularly against drug-resistant cancer cell lines, suggesting their potential as a viable strategy for improving the therapeutic efficacy of frankincense in cancer treatment.

Safeguarding Bee Health: Insights from a Collaborative Monitoring and Prevention Project Against Pesticide Poisonings

In recent decades, bee poisoning due to pesticides and agrochemicals has increased, posing significant challenges to honey bee health and contributing to the so-called “Colony Collapse Disorder” (CCD). Poor knowledge about the level of exposure of bee colonies to pesticides and agrochemicals, whether from the environment or beekeeping management practices, is a major limiting factor in preventing these diseases. Collaboration among different stakeholders, such as beekeeping associations, local veterinary authorities, and researchers, is essential to create monitoring programs that can collect these data and enable the prompt implementation of surveillance and preventive actions to address potential bee colony poisoning incidents caused by these contaminants. The present study describes the results obtained through a collaborative initiative that was implemented for monitoring and preventing pesticide-induced bee poisonings in a territory of northern Italy where the exposure of honey bee colonies to different types of pesticides and agrochemicals may occur. Four sentinel apiaries were selected based on possible sources of pollution throughout the territory. Pollen samples were collected at different times during the years 2021–2022 and analyzed for pesticides using gas and liquid chromatography tandem quadrupole mass spectrometry (GC-MS/MS and LC-MS/MS) techniques. The findings showed the presence of a number of pesticides, such as pyrethroids, permethrin, cypermethrin, amitraz, and pendimethalin, at varying concentrations. Acute bee mortality was observed in a field case related to pyrethroid exposure in 2022, in an urban area. These results confirm the need for timely interventions, improved sampling methods, and continuous monitoring to safeguard bee populations. Collaboration with local beekeepers and public authorities is thus essential in addressing pesticide use and bee health challenges, fostering efficient communication and training efforts to support sustainable beekeeping.

Pulmonary disposition and pharmacokinetics of a single oral dose of chloramphenicol in healthy fasted adult horses

Abstract Objective To describe and compare the pulmonary and plasma pharmacokinetics of different oral formulations of chloramphenicol administered as a single dose to healthy adult horses. Methods A single dose of chloramphenicol was administered to 6 healthy, university-owned fasted adult horses IV (25 mg/kg), orally as commercial tablets (50 mg/kg), or orally or intragastrically as compounded suspension (50 mg/kg), according to a randomized crossover protocol. Plasma was collected 5, 10, 15, 20, and 30 minutes and 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after drug administration. Bronchoalveolar lavage (BAL) fluid was collected after 1, 4, and 8 hours and processed to obtain pulmonary epithelial lining fluid (PELF) and the BAL cell pellet (BALc). Chloramphenicol concentrations were determined by means of liquid chromatography–tandem mass spectrometry in plasma, PELF, and BALc. Data were used for plasma noncompartmental analysis and calculation of apparent PELF and BALc concentrations. Results Chloramphenicol concentrations were higher in the PELF than in plasma, irrespective of formulation and administration route (IV, orally, or intragastrically). Compounded suspension administered intragastrically yielded higher maximum concentration and drug exposure than administered orally, with a relative bioavailability of 79%. After oral administration, no significant differences were found between compounded suspension and commercial tablets. Conclusions Oral administration of chloramphenicol achieved pulmonary concentrations ≥ 2 and 4 µg/mL for at least 4 hours (50% to 75% of a 6- to 8-hour dosing interval) in 4 out of 5 treated horses. Clinical Relevance Pulmonary pharmacokinetics can be used by practitioners to judiciously select an antimicrobial for the treatment of complex equine pneumonia cases.

Widely Targeted Metabolomics Analyses Provide Insights into the Transformation of Active Ingredients During Drying and the Mechanisms of Color Change for Forest Ginseng (Panax ginseng C. A. Mey. cv. Sativi-nemoralis)

In this study, we investigated the mechanism of conversion of active components as well as the color change of forest ginseng (FG) during the drying process with the self-developed negative-pressure circulating airflow-assisted desiccator (PCAD) drying method, using a widely targeted metabolomics analytical method based on ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). During the drying process, a total of 1862 metabolites were identified in FG, along with 748 differential abundant metabolites (DAMs). Further analysis of the types and metabolic pathways of the DAMs revealed that both primary and secondary metabolites changed by 50–70% moisture content (MC); secondary metabolites dominated with a 30–50% MC, and primary metabolites dominated with a 10–30% MC, which revealed the differences in the transformation of the active ingredients in the drying process. In addition, the results showed the browning characteristics during the drying process. MC-50 and MC-10 showed the smallest and largest color changes, as well as enzyme activities, compared to the other MCs, respectively. As drying proceeded, browning reactions were mainly related to lipid and nucleotide metabolism and phenylpropane and flavonoid biosynthesis. In conclusion, the present study provides theoretical support for the mechanisms of active ingredient transformation as well as the color change of FG during PCAD drying.

Gut microbiota interacting with vitamin D but not anandamide might contribute to the pathogenesis of preeclampsia: a preliminary study

IntroductionPreeclampsia (PE) is a pregnancy-specific multisystem disorder and a leading cause of maternal and perinatal mortality globally. Despite numerous studies highlighting the potential roles of gut microbiota, anandamide (AEA), and Vitamin D (VitD) in PE, none have established them as reliable biomarkers for predicting disease onset. Moreover, their interactions in late-stage pregnancy women remain poorly understood.MethodsThirty-four preeclamptic patients (called PE group) and thirty-nine matched healthy late-pregnant women (called LP group) were involved in this case-control study. Fecal samples, which were used to acquire the diversity and composition of gut microbiota, were analyzed by 16S rRNA gene sequencing. Plasma AEA concentrations and serum VitD levels were determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively.ResultsIn this study, β diversity but not α diversity significantly differed between the LP and PE groups. Compared with the LP group, the relative abundances of Prevotella, Erysipelotrichaceae_UCG-003, and Dorea were increased dramatically in the PE group, whereas the relative abundances of Subdoligranulum, Parabacteroides, Bacteroides were significantly decreased in the PE group. Furthermore, women with PE had a substantially lower plasma level of AEA and a marked decrease in serum VitD compared to normal late-pregnant women. Lastly, although the serum level of AEA was not significantly correlated with VitD or any of the top 6 marker genera, VitD was significantly negatively correlated with the relative abundance of Dorea, a novel finding in this context.DiscussionThe gut microbiota profile of the PE group was significantly different from that of the LP group. Although no significant correlations were identified between the plasma AEA levels and serum VitD levels or any of the top 6 identified marker genera, a significant negative correlation was observed between VitD and Dorea, indicating VitD and gut microbiota have the potential to be combined targets for early diagnosis and management of PE.

Development and Validation of a Fast and Sensitive UPLC-MS/MS Method for Ethyl Glucuronide (EtG) in Hair, Application to Real Cases and Comparison with Carbohydrate-Deficient Transferrin (CDT) in Serum

Alcohol is responsible for an ever-increasing number of deaths worldwide, and many road accidents are caused by irresponsible drinking and driving. The use of biomarkers that can support a diagnosis of alcohol abuse is a very important tool that can improve the prevention of many alcohol-related diseases and serious traffic accidents. The main aim of our study was the full validation of a rapid and simple method by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to detect ethyl glucuronide in hair (hEtG). The method was successfully applied to n = 171 real hair samples collected from drivers convicted of driving while impaired by alcohol or drugs. A comparison of hEtG and serum Carbohydrate-Deficient Transferrin percentages (% CDT) was also performed to carefully evaluate the data in relation to the specific detection windows of the two different biomarkers. Most of the drivers with hEtG > 30 pg/mg were males in their thirties. None of the hEtG-positives had a serum % CDT above the cutoff (≥2%). Although some researchers suggest caution until solid data are available on the possible effects of interindividual variability that may influence EtG incorporation and metabolism, hEtG is a very useful biomarker of long-term alcohol exposure that shows greater reliability than traditional blood markers.

Evaluation of Tryptophan and Its Metabolites in Predicting Disease Activation in Inflammatory Bowel Disease

Background and Aim: Inflammatory bowel disease (IBD), which comprises ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by chronic inflammation and fluctuating disease activity. This study aimed to evaluate serum tryptophan (TRP) and its metabolites as potential biomarkers for predicting disease activation in comparison to fecal calprotectin (FC). Methods: This prospective study included 115 patients (77 with UC and 38 with CD). Disease activity was assessed based on clinical and endoscopic findings. Serum TRP levels and their metabolites were measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS), whereas FC levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). Results: Serum TRP levels ≤ 11,328.41 ng/mL predicted disease activation with 72.1% sensitivity and 62.7% specificity, whereas FC levels ≥ 89.60 µg/g showed 84.2% sensitivity and 67.6% specificity. The TRP-to-C-reactive protein (CRP) ratio (TRP/CRP) demonstrated superior diagnostic accuracy, with an area under the curve (AUC) of 0.847. Conclusions: The TRP/CRP ratio is a novel and comprehensive approach for predicting disease activation in IBD patients. Although FC remains the gold standard, TRP and its metabolites provide valuable complementary insights. Further research is required to validate these findings in larger cohorts.

Development and Validation of Targeted Metabolomics Methods Using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) for the Quantification of 235 Plasma Metabolites

Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity and complexity. This study aims to develop and validate targeted LC-MS/MS methods for quantifying 235 mammalian metabolites from 17 compound classes in porcine plasma without prior derivatization. Utilizing reversed-phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, each analyte is identified and quantified using two selected reaction monitoring (SRM) transitions. Fast polarity switching and scheduled SRM enhance the metabolome coverage and throughput, enabling the analysis of one sample in about 40 min. A simple “dilute and shoot” sample preparation protocol was employed, with samples injected at two dilution levels to align metabolite concentrations within calibration curve ranges. Validation in porcine plasma included assessments of carryover, linearity, detection and quantification limits, repeatability and recovery. The method was further applied to plasma samples from various animal species, demonstrating its applicability to human and animal studies. This study establishes two robust LC-MS/MS methods for comprehensive porcine plasma metabolome quantification, advancing large-scale targeted metabolomics in biomedical research.

A novel LC-MS/MS analysis of vitamin D metabolites in mice serum and hair: impact of diet and light exposure

IntroductionNumerous physiological systems, such as the functioning of the immune system, bone health, and the regulation of expression of genes, depend critically on vitamin D. Considering the significance of vitamin D for health, it is critical to understand how it is metabolized and the factors that affect its levels.MethodsThe objective of this study was to develop and validate an LC-MS/MS method to examine the effects of light exposure and dietary vitamin D consumption on the levels of vitamin D and its metabolites in a mouse model under consistent growth conditions throughout the year. Serum and hair samples from mice were analyzed under various experimental conditions for vitamin D and its metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experimental conditions included a vitamin D-deficient diet, a vitamin D-standard diet, and changes in ambient light exposure ranging from complete darkness to a regular light-dark cycle.ResultsMice fed a standard vitamin D diet and exposed to a regular light-dark cycle exhibited significantly higher levels of 25OHD3 in both serum and hair, indicating the synergistic effect of dietary vitamin D intake and light exposure. Mice fed a standard vitamin D diet but kept in continuous darkness showed moderately elevated 25OHD3 levels, demonstrating the efficacy of dietary vitamin D in maintaining adequate levels despite the absence of light. Conversely, mice fed a vitamin D-deficient diet and housed in darkness displayed 25OHD3 levels below the limit of quantification, highlighting the combined detrimental effects of dietary deficiency and lack of light exposure.DiscussionThis study provides valuable insights into the complex interplay between dietary vitamin D intake, light exposure, and the regulation of vitamin D metabolism in mice. Moreover, our results underscore the potential implications for human health, suggesting the importance of adequate vitamin D intake and sunlight exposure in maintaining optimal vitamin D levels. Further research in this area has the potential to unveil additional factors influencing vitamin D metabolism, offering valuable insights into strategies for optimizing vitamin D levels in both animal models and human subjects.

Quantification of mycophenolic acid in plasma by isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method.

Accurate measurements of plasma mycophenolic acid (MPA) are essential for therapeutic drug monitoring in transplant recipients and autoimmune diseases. The performance of plasma mycophenolic acid routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of plasma mycophenolic acid measurements. In this study, sample preparation was based on protein precipitation with methanol followed by further dilution. The mycophenolic acid was quantified by the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with electrospray ionization in positive ion mode. According to the Clinical and Laboratory Standards Institute (CLSI) documents C62-A and C50-A, the basic analytical performance of the candidate reference method was verified, such as linearity, limit of quantification, matrix effect, precision, accuracy, and uncertainty. Moreover, the candidate reference measurement procedure was compared with the routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a clinical laboratory. Based on the data, the mycophenolic acid in human plasma was well detected by ID-LC-MS/MS. No apparent interferences were found with the mycophenolic acid measurement. The calibration curve for the mycophenolic acid was linear in the concentration range of 0.1-50μg/mL with a correlation coefficient of 0.9999 under the optimum experimental conditions. This method was sensitive because the low limit of quantitation (LOQ) was 0.05μg/mL. The recoveries of MPA were 98.11-98.95%. The intra-day and inter-day coefficients of variations (CV) of our method were ≤ 1.53% and ≤ 0.51%, respectively. No obvious matrix effect was observed. There was a good correlation between this method and the clinical routine LC-MS/MS method. To sum up, we established and validated a reliable plasma MPA method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for plasma MPA routine measurements.

Combined metabolomics and 16S rDNA sequence analyses of the gut microbiome reveal the action mechanism of Fructus Akebiae against hepatic fibrosis

ObjectivesTo explore the mechanism underlying the effect of Fructus Akebiae (FAE) against hepatic fibrosis in mice through combined network pharmacology, liver metabolomics, and 16S rDNA analyses of the gut microbiota.MethodsIn this study, we randomly divided mice into the control, model, FAE high-dose, FAE medium-dose, and FAE low-dose groups to analyze the pathological changes in the hepatic fibrosis and levels of the α-SMA, collagen 1, Nuclear Factor Kappa B (NF-κ B), Toll Like Receptor 4 (TLR4). The gut microbiota was analyzed through 16S rDNA sequencing analysis of liver metabolites using liquid chromatography-mass spectrometry. Furthermore, network pharmacology was used to determine the specific molecular regulation mechanism of FAE in hepatic fibrosis treatment.ResultsFAE treatment markedly improved the pathological changes in the hepatic fibrosis. Analysis revealed that FAE administration reversed the carbon tetrachloride (CCl4)-induced dysbiosis by increasing the abundance of Akkermansia and reducing that of Cyanobacteria. Additionally, metabolomic analysis showed that FAE treatment reversed the CCl4-induced metabolic disorders by regulating amino and nucleotide sugar metabolism. Furthermore, correlation analysis showed that Akkermansia and Verrucomicobiota were closely related to D-tolasaccharide and maltotetraose saccharide. Moreover, network pharmacology indicated that FAE might regulate the signaling pathway through the JUN/CASP3/NOS3/PTGS2/HSP90AA1 during treatment.ConclusionFAE may be a promising treatment for hepatic fibrosis, and its protective effects are associated with improvements in the microbiome and metabolic disorders.

Two-Photon Direct Laser Writing of Capillary-Coupled Nanosprayer Arrays for ESI-MS.

Electrospray is a widely recognized physical process and has various applications, such as in electrospinning, space propulsion, and mass spectrometry (MS). Particularly, electrospray emitters are integral to the exceptional performance of electrospray ionization mass spectrometry (ESI-MS). This study introduced a novel method for fabricating electrospray emitters using two-photon direct laser writing, resulting in the printing of nanosprayer arrays directly onto capillary tips. Utilizing a novel acrylic polyhedral oligomeric silsesquioxane photoresist, a multinozzle sprayer with micrometer scale channels was designed and printed on a capillary that is compatible with both frontend liquid chromatography and typical MS inlets. The 3D-printed sprayers demonstrated exceptional resistance to organic corrosion and high temperatures. For the nanosprayer arrays, an increase in the total electrospray current was achieved by augmenting the number of electrosprays, while maintaining a constant liquid flow rate and voltage. Notably, the eight-nozzle sprayers exhibited a 2.7-fold enhancement in sensitivity as compared with single-nozzle sprayers. This approach offers a versatile and high-sensitivity nanoelectrospray solution for ESI-MS.

Investigation on Rational Utilization of Medicinal Plant Semiliquidambar cathayensis Chang Leaf and Bark at Different Developmental Stages

Background:Semiliquidambar cathayensis Chang is an extremely valuable and endangered medicinal plant. To investigate the exploitation and rational utilization of S. cathayensis, this study conducted metabolomics analysis of the leaves and bark of artificially cultivated S. cathayensis at different developmental stages. Methods: These metabolites were detected and identified by Ultra-Performance Liquid Chromatography–tandem Mass Spectrometry (UPLC-MS/MS) technology, and then univariate statistical analyses, multivariate pattern analyses, and pathway analyses were carried out. Results: As a result, a total of 801 metabolites were detected in S. cathayensis; differential metabolites in leaves at different developmental stages were mainly enriched in pathways related to flavonoids, whereas differential metabolites in bark at different developmental stages were mainly aromatic compounds, amino acids, and flavonoids, among others. This study revealed that young leaves are ideal for use in treating rheumatism, regulating blood pressure, and lowering blood glucose, while old leaves are better suited for skincare products and extracting materials to prevent neurodegenerative diseases and support women’s ovarian health. As for bark, four-year-old S. cathayensis bark is optimal for extracting myricetin. If the pharmaceutical, chemical, food, and industrial fields require extensive extraction of L-phenylalanine, trans-3-hydroxycinnamate, and 4-hydroxyphenylacetate, and if the medical field needs to extract anti-allergy, liver protection, and anti-coagulant ingredients, the two-year-old S. cathayensis bark is the best choice. Conclusions: Thus, this study established a solid theoretical framework for the rational, effective, and sustainable utilization of S. cathayensis leaves and bark.

Spatial Proteomics by Parallel Accumulation-Serial Fragmentation Supported MALDI MS/MS Imaging: A First Glance Into Multiplexed and Spatial Peptide Identification.

In spatial proteomics, matrix-assisted laser desorption/ionization (MALDI) imaging enables rapid and cost-effective peptide measurements. Yet, insitu peptide identification remains challenging. Therefore, this study aims to integrate the trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) into MALDI imaging of tryptic peptides to enable multiplexed MS/MS imaging. An initial MALDI TIMS MS1 survey measurement was performed, followed by a manual generation of a precursor list containing mass over charge values and ion mobility windows. Inside the dual TIMS system, submitted precursors were trapped, separately eluted by their ion mobility and analyzed in a quadrupole time-of-flight device, thereby enabling multiplexed MALDI MS/MS imaging. Finally, precursors were identified by peptide to spectrum matching. This study presents the first multiplexed MALDI TIMS MS/MS imaging (iprm-PASEF) of tryptic peptides. Its applicability was showcased on two histomorphologically distinct tissue specimens in a four-plex and five-plex setup. Precursors were successfully identified by the search engine MASCOT in one single MALDI imaging experiment for each respective tissue. Peptide identifications were corroborated by liquid-chromatography tandem mass spectrometry experiments and fragment colocalization analyses. In this study, we present a novel pipeline, based on iprm-PASEF, that allows the multiplexed and spatial identification of tryptic peptides in MALDI imaging. Hence, it marks a first step towards the integration of MALDI imaging into the emerging field of spatial proteomics.

MYSM1 Mediates Cardiac Parthanatos and Hypertrophy by Deubiquitinating PARP1.

Cardiac hypertrophy constitutes the primary pathological basis for heart failure and exerts a considerable influence on morbidity and mortality. Deubiquitinating enzymes are crucial regulators of protein degradation and play a pivotal role in cardiac pathophysiology. This study aimed to clarify the involvement of a deubiquitinating enzyme, MYSM1 (Myb-like, SWIRM, and MPN domains 1), in cardiac hypertrophy and to explore the underlying mechanism. Cardiac hypertrophy was developed by angiotensin II or transverse aortic constriction surgery. Cardiomyocyte-specific knockdown of MYSM1 was accomplished using the adeno-associated virus serotype 9-cTNT-Mysm1-shRNA. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry analysis was utilized to identify potential substrates of MYSM1. First, we discovered that the expression of MYSM1 increases during cardiac hypertrophy. MYSM1 knockdown mitigated cardiac dysfunction and hypertrophy after angiotensin II administration. Cardiomyocyte-specific knockdown of MYSM1 with adeno-associated virus serotype 9 alleviated myocardial dysfunction and hypertrophy caused by transverse aortic constriction surgery. Through co-immunoprecipitation and LC-MS, poly (ADP-ribose) polymerase 1 (PARP1) was identified as a potential substrate protein of MYSM1. PARP1 initiates a novel form of programmed cell death termed parthanatos, which is characterized by excessive PARylation, nuclear translocation of AIF, and depletion of nicotinamide adenine dinucleotide. MYSM1 deubiquitinates and stabilizes PARP1 in an MPN domain-dependent manner. In addition, MYSM1 mediates cardiac hypertrophy through PARP1-dependent cardiomyocyte parthanatos. This study identified the role of the MYSM1-PARP1 axis in mediating cardiac hypertrophy and suggested that MYSM1 is a promising pharmacological target for the treatment of cardiac hypertrophy.

The effect of storage time and temperature on the proteomic analysis of FFPE tissue sections

Formalin-fixed paraffin-embedded (FFPE) tissues present an important resource for cancer proteomics. They are more readily available than fresh frozen (FF) tissues and can be stored at ambient temperature for decades. FFPE blocks are largely stable for long-term preservation of tumour histology, but the antigenicity of some proteins in FFPE sections degrades over time resulting in deteriorating performance of immunohistochemistry (IHC). It is not known whether FFPE sections that have previously been cut from blocks and used for liquid chromatography-mass spectrometry (LC–MS) analysis at a later time are affected by storage time or temperature. We determined the stability of FFPE sections stored at room temperature (RT) versus − 80 °C over 48 weeks. The stored sections were processed at different timepoints (n = 11) and compared to sections that were freshly cut from FFPE blocks at each timepoint (controls). A total of 297 sections (rat brain, kidney and liver stored at RT, − 80 °C or freshly cut) were tryptically digested and analysed on TripleTOF 6600 mass spectrometers in data-dependent acquisition (DDA) mode. Kidney and liver digests were also analysed in data-independent acquisition (DIA) mode. The number of proteins and peptides identified by DDA with ProteinPilot and some common post-translational modifications (PTMs) were unaffected by the storage time or temperature. Nine of the most common FFPE-associated modifications were quantified using DIA data and all were unaffected by storage time or temperature. Therefore, FFPE tissue sections are suitable for proteomic studies for at least 48 weeks from the time of sectioning.

A Bioanalytical Liquid Chromatography Tandem Mass Spectrometry Approach for the Quantification of a Novel Antisense Oligonucleotide Designed for Parkinson’s Disease: A Rat Brain Biodistribution Study

A Bioanalytical Liquid Chromatography Tandem Mass Spectrometry Approach for the Quantification of a Novel Antisense Oligonucleotide Designed for Parkinson’s Disease: A Rat Brain Biodistribution Study