Multiple Reaction Monitoring Method Research Articles

Determination of the extremolyte ectoine in plasma and a pharmacokinetic study in rats by a validated and BAGI-evaluated UPLC-MS/MS method

Ectoine (ECT) has recently gained considerable interest in the healthcare sector due to its promising therapeutic benefits in a variety of human disorders. This research aimed to quantify the ECT plasma level in rats by creating and optimizing a sensitive and validated UPLC-MS/MS method. Prior to analysis, ECT extraction from the plasma samples was conducted via a protein precipitation procedure, using hydroxyectoine as an internal standard (IS). A 1.7 μm UPLC C8 column (100 mm × 2.1 mm) was selected for the chromatographic separation, using a gradient mobile phase consisting of acetonitrile and 0.05% formic acid. The electrospray ionization mass spectrometry (ESI-MS) was used to detect ECT in the positive ion mode. To determine the specific precursor and the product ions of ECT, multiple reaction monitoring (MRM) methods were carried out. The selected ion pair of ECT was 143.1 > 97 and 159.1 > 113.13 for the IS. The ECT’s linearity range in rat plasma was found to be 1-1000 ng/mL, with a recovery rate of 96.48–97.37%. Consistent with FDA guidelines for bio-analytical method validation, the suggested method was validated. The method was efficiently employed to quantify the studied drug in spiked rat plasma with good accuracy and precision with no significant matrix effects. Furthermore, it was effectively used to investigate the pharmacokinetic behavior of ECT in rats after a single oral dose of 30 mg/kg.

B-288 Turning False Positive Screening into True Positive Results

Abstract Background After conducting a retrospective analysis of our laboratory data, we identified a notable disparity between immunoassay screen and mass spectrometry confirmation results for benzodiazepines. This prompted an investigation into whether the prevalence of false negative benzodiazepine results stemmed from the inadequacy of the immunoassay or the absence of new psychoactive substances (NPS) in our mass spectrometry confirmation assay. To determine the latter, we added four NPS benzodiazepines and their metabolites to our confirmation panel. Monitoring drug seizure records as well as reports detailing emerging substances were helpful in guiding which NPS to add. The objective of this study was to determine whether the addition of NPS benzodiazepines to our confirmation panel would increase the agreement between screen and confirmation benzodiazepine results in an opioid dependent population. Methods Urine samples that screened positive for benzodiazepines with immunoassay and confirmed negative by mass spectrometry over a 6-month timeframe were selected for analysis. Four NPS were added to our confirmation panel which included bromazolam, flubromazepam, flubromazolam, flualprazolam as well as their metabolites alpha-OH bromazolam, 3-OH flubromazepam, alpha-OH flubromazolam and alpha-OH flualprazolam. Immunoassay data was obtained using an Olympus AU480 instrument, while mass spectrometry results were acquired through an internally developed dynamic multiple reaction monitoring method on an Agilent 6470 triple quadrupole instrument. Results In the analysis of 476 urine samples using a cut-off value of 50 ng/mL, 55.3% of the samples tested positive for bromazolam, 83.0% for alpha-OH bromazolam, 0% for flubromazepam, 41.8% for 3-OH flubromazepam, 0% for flubromazolam, 0.2% for alpha-OH flubromazolam, 1.3% for flualprazolam and 6.7% for alpha-OH flualprazolam. Using a cut-off value of 10 ng/mL which represents the limit of quantitation, 75.4% tested positive for bromazolam, 89.9% for alpha-OH bromazolam, 2.1% for flubromazepam, 60.5% for 3-OH flubromazepam, 0% for flubromazolam, 0.2% for alpha-OH flubromazolam, 3.4% for flualprazolam and 9.7% for alpha-OH flualprazolam. In total, among the 476 samples that initially screened positive but confirmed negative, 95.8% confirmed positive for benzodiazepines with the inclusion of the four NPS benzodiazepines and their metabolites into our confirmation panel. Of the samples that tested positive for NPS benzodiazepines, 77.1% were also positive for fentanyl, and 95.2% were positive for norfentanyl. Conclusions The incorporation of NPS benzodiazepines into our confirmation panel significantly enhanced the concordance between immunoassay screen and mass spectrometry confirmation results. The discrepancies we observed with benzodiazepines were not predominantly linked to immunoassay challenges. Rather, they were due to the presence of NPS benzodiazepines commonly found in benzo-dope preparations, not accounted for in our mass spectrometry confirmation panel. The inclusion of NPS benzodiazepines into our confirmation panel has improved the reporting of useful diagnostic information which can be used to support harm reduction efforts.

Targeted LC-MS/MS method of oxylipin profiling reveals differentially expressed serum metabolites in type 2 diabetes mice with panaxynol

Panaxynol is a bioactive polyacetylene in food plants; however, its specific benefits in diabetes and metabolic disorders remain unclear. Previous studies have mainly focused on biochemical indicators and clinical evaluations. Limited research has systematically elucidated the beneficial effects of panaxynol from the oxylipins perspective. In this study, we employed an oxylipin analysis platform we previously established using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based on the multiple reaction monitoring (MRM) method for the profiling of oxylipins. After a 7-week administration of panaxynol to db/db mice, significant alterations in serum oxylipins and potential benefits to hyperglycemia, insulin resistance, and hepatic steatosis were observed. Our analysis also revealed correlations among epoxygenase products derived from arachidonic acid (AA), linoleic acid (LA), and α-linolenic acid (ALA) via cytochrome P450 (CYP) pathways. Furthermore, six potential oxylipins were identified, as offering insights into the mechanisms by which panaxynol may modulate diabetes. These results provide the first in vivo evidence of the impact of panaxynol on oxylipin metabolism and lay the foundation for developing panaxynol as a nutraceutical for diabetes management.

An economically viable stable isotope-enhanced multiple reaction monitoring method for total fatty acid analysis in a mouse model of non-alcoholic fatty liver disease

The complex pathological mechanisms of non-alcoholic fatty liver disease (NAFLD) are closely related to dysregulated lipid metabolism, and the therapeutic effects of the traditional Chinese medicine Zexie-Baizhu Decoction (AA) on NAFLD have been gaining increasing attention. However, research into altered lipid metabolism, especially fatty acids, in NAFLD and the intervention of AA faces technical challenges, especially in the precise quantitative analysis of fatty acids in biological samples. The high complexity of biological matrices, particularly after drug intervention, greatly increases the difficulty of detection. Therefore, this study innovatively developed a simple and economical stable isotope derivatization technique by synthesizing d6N,N-dimethylethylenediamine (d6-DMED) in the laboratory, establishing a simple and economical method for fatty acid quantification. This method employs a chemical reaction under low-temperature conditions to ensure the efficient synthesis of d6-DMED. Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technique (UHPLC-MS/MS), combined with optimized chromatographic separation conditions and dynamic multiple reaction monitoring mode, the study established a highly sensitive detection method for 35 fatty acid derivatives. Methodological evaluation showed that the limits of quantification ranged from 0.002 to 0.060 μM, with high linearity of R² > 0.995. Additionally, the relative recovery rates were between 93.14% and 106.63%. To further demonstrate the feasibility of this method for fatty acid quantification, it was applied to measure fatty acids in multiple tissues in a mouse NAFLD model, as well as the effects of AA intervention on fatty acid metabolism. This rapid, simple, and cost-effective detection method not only enhances the understanding of NAFLD mechanisms but also provides a new strategy for evaluating the biological complex system after drug intervention.

Contactin proteins in cerebrospinal fluid show different alterations in dementias.

The proteins contactin (CNTN) 1-6 are synaptic proteins for which there is evidence that they are dysregulated in neurodegenerative dementias. Less is known about CNTN changes and differences in cerebrospinal fluid (CSF) of dementias, which can provide important information about alterations of the CNTN network and be of value for differential diagnosis. We developed a mass spectrometry-based multiple reaction monitoring (MRM) method to simultaneously determine all six CNTNs in CSF samples using stable isotope-labeled standard peptides. The analytical performance of the method was evaluated for peptide stability, dilution linearity and precision. CNTNs were measured in 82 CSF samples from patients with Alzheimer's disease (AD, n = 19), behavioural variant frontotemporal dementia (bvFTD, n = 18), Parkinson's disease dementia/dementia with Lewy bodies (PDD/DLB, n = 18) and non-neurodegenerative controls (n = 27) and compared with core AD biomarkers. The MRM analysis revealed down-regulation of CNTN2 (fold change (FC) = 0.77), CNTN4 (FC = 0.75) and CNTN5 (FC = 0.67) in bvFTD and CNTN3 (FC = 0.72), CNTN4 (FC = 0.75) and CNTN5 (FC = 0.73) in PDD/DLB compared to AD. CNTN levels strongly correlated with each other in controls (r = 0.73), bvFTD (r = 0.86) and PDD/DLB (r = 0.70), but the correlation was significantly lower in AD (r = 0.41). CNTNs in AD did not show correlation even with core AD biomarkers. Combined use of CNTN1-6 levels increased diagnostic performance of AD core biomarkers. Our data show CNTNs differentially altered in dementias and indicate CNTN homeostasis being selectively dysregulated in AD. The combined use of CNTNs with AD core biomarkers might help to improve differential diagnosis.

Targeted lipidomics-based study of radiation-induced metabolite profiles changes in plasma of total body irradiation cases

Purpose Lipidomics is an important tool for triaging exposed individuals, and helps early adoption of prevention and control strategies. The purpose of this study was to screen significantly perturbed lipids between pre- and post-irradiation of human plasma samples after total body irradiation (TBI) and explore potential radiation biomarkers for early radiation classification. Methods Plasma samples were collected before and after irradiation from 22 hospitalized cases of acute myeloid leukemia (AML) prepared for bone marrow transplantation. Acute total-body γ irradiation was performed at doses of 0, 4, 8, and 12 Gy. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring (MRM) method was utilized. Self-paired studies before and after irradiation were performed to screen potential lipid categorization markers and markers of dose-response relationships for radiation perturbation in humans. Based on the screened potential markers, a human TBI dose estimation model was developed. Results In total, 426 individual lipids from 14 major classes were quantified and 152 potential biomarkers with categorical characteristics were screened. A total of 80 lipids (32 TGs, 29 SMs, 9 FAs, 5 CEs, 5 PIs) were upregulated at 4 Gy, and a total of 91 lipids (39 SMs, 18 TGs, 15 HexCers, 7 CEs, 6 Cers, 3 LacCers, 2 LPEs, 1 PI) were upregulated at 12 Gy. Comparison of the ROC curves between the non-exposed and exposed groups at different doses showed AUC values ranging from 0.807 to 0.876. The metabolic pathways of potential lipid markers are mainly sphingolipid and glycerolipid metabolism, unsaturated fatty acid biosynthesis, fatty acid degradation and biosynthesis. Among the 13 dose-dependent radiosensitive lipids, CE (20:5), CE (18:1) and PI (18:2/18:2) were gradually incorporated into the TBI dose estimation model. Conclusion This study suggested that it was feasible to acquire quantitative lipid biomarker panels using targeted lipidomics platforms for rapid, high-throughput triage. Lipidomics strategies for radiation biodosimetry in humans were established with lipid biomarkers with good dose-response relationship.

Lipidomics analysis of bone marrow in a mouse model of postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP) is a major public health problem worldwide, afflicting many postmenopausal women. Although many studies have focused on the biological role of individual lipids in osteoporosis, no studies have systematically elucidated the lipid profile of osteoporosis. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology based on multiple reaction monitoring (MRM) method was used to compare the levels of lipid molecules in bone marrow cells of osteoporotic mice (OVX) group and sham-operation (Sham) group. Principal component analysis (PCA) was used for multivariate statistics. Differential lipids were obtained by bar graph, heatmap and volcano map. A total of 400 lipid molecules were identified. A total of 199 lipid molecules were identified to be associated with PMOP, including 6 phospholipids and 3 sphingolipids. These differential lipid molecules provide a systematic lipid profile for osteoporosis, which helps to discover new candidate osteoporosis biomarkers, and their changes at the molecular level can be used as new targets for diagnosis or prevention.

Integrated identification-quantification (ID-Quant) workflow utilizing UPLC-QTOF-MS for the therapeutic drug monitoring of multi-component antibiotics without pure standards: Validation using teicoplanin

The lack of individual pure standard has hampered the application of therapeutic drug monitoring (TDM) for multi-component antibiotics in clinical laboratories. Here, we aimed to develop an integrated identification-quantification (ID-Quant) workflow based on ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) to enable the comprehensive determination of all teicoplanin components without needing pure standards. The workflow comprises three steps. First, non-targeted MSE full scanning was used to detect and identify all potential ingredients. Then, characteristic product ions were selected to generate a quantitative time-of-flight multiple reaction monitoring (Tof-MRM) method. Finally, the constituent composition of teicoplanin injection was determined and utilized as an alternative reference standard to monitor the teicoplanin ingredients in human serum samples. As a result, nine teicoplanin analogs were identified from teicoplanin injection (Sanofi-Aventis, France). The overall performance of the Tof-MRM method was satisfactory in terms of linearity, precision, accuracy, and limits of detection. Utilizing the drug as standard, the individual concentrations for each component in patient serum were determined to be 0.120 µg/mL (A3-1), 0.020 µg/mL (N-1), 0.550 µg/mL (N-2), 0.730 µg/mL (A2-1), 4.26 µg/mL (A2-2,3), 4.79 µg/mL (A2-4,5), and 0.290 µg/mL (N-3), respectively. The distribution pattern of teicoplanin components was also discovered to differ from that in the drug injection. Overall, this integrated ID-Quant workflow based on UHPLC-QTOF-MS enables the robust quantitation of all teicoplanin analogs without the need for individual pure standard. This approach could help address the standard unavailability problem in the TDM of multi-component antibiotics.

The bone marrow lipidomics of mice reveal sex-related differences.

Osteoporosis is a common skeletal disorder characterized by an imbalance between bone resorption and formation, exhibiting a higher prevalence in women compared with men. While previous studies have primarily focused on genomics and genetics in osteoporosis susceptibility, there is a lack of systematic exploration of sex-specific differences in lipid levels in mouse bone marrow. Multiple reaction monitoring-based liquid chromatography-trandem mass spectrometry(LC-MS/MS) was used to quantify lipidomic profiles in bone marrow samples from three female mice and three male mice. The LC-MS/MS technique based on the multiple reaction monitoring method identified and quantified 184 lipids from 15 lipid classes. The contents of most lipids in the bone marrow cells of female mice were higher than those in male mice, including four polyunsaturated fatty acids, three phospholipids and four sphingolipids. Among all the lipid molecules, lactosylceramide (d18:0/16:0) showed the highest fold change in female mice, while its precursor lipid, glucosylceramide, was the most up-regulated in male mice. This study, focusing on bone marrow lipidomics, elucidates significant sexual dimorphism in lipid levels within bone marrow cells. It provides novel evidence supporting the higher prevalence of osteoporosis in women and enhances our understanding of the connection between sex-specific lipid levels and the risk of osteoporosis.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

Co-stimulatory pathway competitive assay development using Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Discovery and identification of interspecies peptide biomarkers in the seahorse species using liquid chromatography tandem mass spectrometry and chemometrics

Seahorses have important edible and medicinal values including strengthening the body, tonifying the liver and kidneys, and reducing swelling. And there are abundant seahorse species on Earth. Many seahorses have large price differences due to the scarcity of resources, and some seahorses with similar appearances appear to be confused for use. While in market trading, Hippocampus is susceptible to loss of specialized morphology characteristics, making it difficult to distinguish between specific species. Here we report an effective method based on peptide biomarkers for the identification of seahorse species. Peptide biomarkers for each species were predicted using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with chemometrics software. One unique biomarker peptide for each species was synthesized and verified, and finally developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) multiple reaction monitoring method. The results indicate that the method has great potential for species-specific identification of seahorses and their preparations, among others.

Optimization of therapeutic drug monitoring of vancomycin in newborns using “Dried Blood Spot” method

Therapeutic drug monitoring (TDM) is used to increase the individualization of pharmacotherapy, especially in patient groups with a high interindividual variability in pharmacokinetic (PK) parameters. One of these groups of patients is newborn children, for whom drug therapy, especially drugs with a narrow therapeutic range, causes a few difficulties or cannot be used in principle.The aim of the work was to develop and validate quantitative HPLC-MS/MS methods for the determination of vancomycin in “dried blood spot” samples using new protocols and comparison of the results obtained with the results in plasma samples using standard sample preparation methods.Materials and methods. To prepare stock and standard solutions of vancomycin and norvancomycin as an internal standard, dry portions of the corresponding certified standards of vancomycin (Servier, France) and norvancomycin (Augsburg, Germany, purity grade >95.0%) were used. A chromatographic separation of the components was carried out on a Poroshell 120 C18 column (4.6×50 mm, 2.7 µm). When developing conditions for a mass spectrometric detection of the desired substances using the multiple reaction monitoring (MRM) method, precursor ions and their corresponding product ions were determined.Results. A quantitative HPLC-MS/MS method for the determination of vancomycin in «dried blood spot» samples was developed and validated. A comparison was made between vancomycin concentrations in «dried blood spot» samples and plasma samples. Moreover, more than 95% of the calculated average concentrations are within the limits of d-2s and d+2s, which correspond to the values of –10.2 and 12.2. That confirms the suitability of the developed method for the analysis of patient samples.Conclusion. The results obtained make it possible for us to recommend the “dried blood spot” method for therapeutic monitoring of vancomycin, additional studies of PK in this group of patients with subsequent use of this drug in newborns and pediatric patients.

A candidate reference measurement procedure for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring.

α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav1.1, Nav1.2, and Nav1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

RationaleNav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development.MethodsUnique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry.ResultsNav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.ConclusionsMultiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 ≫ Nav1.1 > Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Rapid detection of heat stress biomarkers in Atlantic salmon (Salmo salar) liver using targeted proteomics

AbstractMost fish are ectothermic; therefore, their physiology is significantly affected by temperature. Aquaculture fish have limited ability to avoid elevated water temperatures, with impacts increasing as a result of climate change. To date, quantifying gene expression has been proposed to monitor heat stress in salmon liver. This study aimed to establish a faster multiplexed proteomics method to measure the abundance of thermal stress biomarkers in liver of salmon reared at 15°C or 20°C. Moreover, this study aimed to determine the effects that sample pooling, and data normalisation using housekeeping (HK) protein peptides would exert over the statistical significance of these thermal stress markers. A multiple reaction monitoring (MRM) mass spectrometry method, comprised 45 peptides derived from thermal stress markers and 10 peptides from HK proteins, was applied to measure these markers in liver of salmon reared at 15°C or 20°C. When samples were processed individually, 34 peptides were significant between salmon livers at 15°C or 20°C. In pooled samples, this decreased to five significant peptides. Peptides hprt1_HYADDLDR (hypoxanthine phosphoribosyl transferase) and gapdh_VPTPNVSVVDLTVR (glyceraldehyde‐3‐phosphate dehydrogenase) were the most stable and unstable HK protein peptides, respectively. When data was normalised with hprt1_HYADDLDR, 16 peptides were significant in individual samples and 13 in pooled samples. Significant peptides serpinh1a_ADLSNISGK, SerpinH1_TNSILFIGR, ela2_VVGGEDVR and gapdh_VPTPNVSVVDLTVR were common regardless of data strategy. A fast and reliable MRM method was established to validate thermal stress markers in salmon liver, where individual samples yielded better results than pooled samples. Sample pooling was only better when combined with normalisation as it validated twice the number of markers than sample pooling alone. This method could be applied to monitoring stress response in experiments involving feeding additives designed to mitigate thermal stress or in selective breeding programs to help understanding family variance in thermal tolerance.

Exploring peptides from toad venom for source identification by LC-MS/MS using MRM method

Toad venom is a traditional Chinese medicine (TCM) with various sources and wide-ranging preparations. Previous quality assessment studies primarily concentrated on small molecular compounds like toad dienolactones and indole alkaloids, studies on macromolecular peptides and proteins as quality assessment standards remained at the qualitative stage, lacking the development of practical and convenient quantitative methods. In this study, to explore the peptides from toad venom as a new method for identifying and evaluating its source, a complete scan of the water extract of peptides from toad venom was conducted using HPLC-Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF) 5600, leading to the identification of peptides based on mass spectrometry data. Subsequently, HPLC- Quadrupole-Linear Ion Trap Mass Spectrometer (Q-Trap) 5500 employing Multiple Reaction Monitoring (MRM) mode was utilized to quantitatively analyze peptides in various sources of toad venom, followed by Partial Least Squares Discriminant Analysis (PLS-DA) to further analyze the data and evaluate the effectiveness. This study highlights the importance of exploring macromolecular substance in natural products research and provides a foundation for further studies on toad venom.

Comprehensive analysis of different types of ginsenosides in the different parts of American ginseng by targeted and nontargeted MS/MS scanning.

To comprehensively study the ginsenosides distribution in the various tissues of American ginseng, the qualitative and quantitative-targeted and nontargeted mass spectroscopic methods were established using the high-performance liquid chromatography coupled with Qtrap triple quadrupole mass spectrometry (HPLC-QtrapQQQ-MS). The total ginsenosides of the root, stem, and leaf of American ginseng were determined by a colorimetric method, and the contents showed the order from high to low root, stem, and leaf. Eighty-two kinds of ginsenosides were detected in the different parts of American ginseng by enhanced mass scan-information-dependent data acquisition (IDA)-enhanced product ion (EPI) scan mode, including 69 from the root, 62 from the stem, and 48 from the leaf. An HPLC-multiple reaction monitoring (MRM) method was established, and 28 representative ginsenosides were further quantified in the three parts. Nearly all ginsenosides had the highest contents in the root and the lowest content in the leaf. Three types of ginsenosides (protopanaxadiol [PPD]-, protopanaxatiol [PPT]-, and oleanolic acid [OA]-types) were analyzed by precursor ion-IDA-EPI and MRM-IDA-EPI scan modes. Root had the most abundant ginsenosides in PPD- and PPT-type ginsenosides. Meanwhile, the OA-type ginsenosides are significantly enriched in the stem and leaf of American ginseng. The results provided a supplement to the quality assessment of American ginseng. PRACTICAL APPLICATION: The distribution profile of ginsenosides in the parts of American ginseng is different. Except for the root, the stem, and leaf of American ginseng have the most abundant ginsenosides in oleanolic acid type. The results reported herein can help the manufacturers choose appropriate materials to extract the ginsenosides.

Effect of oxyresveratrol on the quality and membrane lipid metabolism of shiitake mushroom (Lentinus edodes) during storage.

The effect of oxyresveratrol on postharvest quality and membrane lipid metabolism of shiitake mushroom was investigated. The result exhibited that oxyresveratrol retarded browning, maintained firmness and alleviated occurrence of decay of shiitake mushroom. The oxidation and hydrolysis of membrane phospholipids were suppressed by oxyresveratrol treatment, which was associated with reduced LOX and PLD activities and increased SOD and CAT activities. The membrane lipidomics of shiitake mushroom was determined by LC-MS. 385 lipid species and 13 fatty acids in membrane lipids were identified by multiple reaction monitoring method. Compared with control group, the phospholipic acid and lysophospholipid reduced by 29.24% and 21.29% in oxyresveratrol-treated group, respectively, which alleviated hydrolysis of phospholipid. Meanwhile, oxyresveratrol maintained the unsaturation of fatty acids and alleviated oxidation of phospholipid. These results demonstrated that oxyresveratrol could play a dual role of inhibiting the oxidation and hydrolysis of phospholipids to mitigate cellular damage of shiitake mushroom.

Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry for Simultaneous Analysis of 22 Analytes of Oncheong-Eum, a Traditional Korean Herbal Formula

Oncheong-eum (OCE) is a traditional Korean herbal formula comprising eight medicinal herbs for treating skin disorders, including eczema and skin rashes. Here, we sought to simultaneously analyze 22 analytes of OCE using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). All analytes were separated on a Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) maintained at 45 °C by gradient elution with a mobile phase of 0.1% (v/v) aqueous formic acid–acetonitrile. By applying a multiple reaction monitoring method, we rapidly determined the various analytes simultaneously. The coefficient of determination of the regression equation prepared in the tested concentration range of each authentic reference standard was ≥0.9950 and showed good linearity. The accuracy ranged from 84.23% to 115.47%, and the relative standard deviation values for intra- and interday precisions ranged from 0.84% to 9.57%, respectively. Analysis of OCE samples using this method showed that they contained up to 27.10 mg/g of active ingredients. The method can provide data to improve the consistency and, thus, the future quality of OCE preparations and other traditional herbal formulas.