Liquid Chromatography-multiple Reaction Monitoring Research Articles

Exploration of the Amino Acid Metabolic Profiling and Pathway in Clonorchis sinensis-Infected Rats Revealed by the Targeted Metabolomic Analysis.

Background: Clonorchiasis remains a serious public health problem. However, the molecular mechanism underlying clonorchiasis remains largely unknown. Amino acid (AA) metabolism plays key roles in protein synthesis and energy sources, and improves immunity in pathological conditions. Therefore, this study aimed to explore the AA profiles of spleen in clonorchiasis and speculate the interaction between the host and parasite. Methods: Here targeted ultrahigh performance liquid chromatography multiple reaction monitoring mass spectrometry was applied to discover the AA profiles in spleen of rats infected with Clonorchis sinensis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (KEGG) was performed to characterize the dysregulated metabolic pathways. Results: Pathway analysis revealed that phenylalanine, tyrosine, and tryptophan biosynthesis and β-alanine metabolism were significantly altered in clonorchiasis. There were no significant correlations between 14 significant differential AAs and interleukin (IL)-1β. Although arginine, asparagine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were positively correlated with IL-6, IL-10, tumor necrosis factor (TNF)-α as well as aspartate aminotransferase and alanine aminotransferase; β-alanine and 4-hydroxyproline were negatively correlated with IL-6, IL-10, and TNF-α. Conclusion: This study reveals the dysregulation of AA metabolism in clonorchiasis and provides a useful insight of metabolic mechanisms at the molecular level.

Abstract 5925: Safety, efficacy, pharmacokinetics, and pharmacodynamics of a second generation Irofulven analog CAP-0121 in preclinical cancer models

Abstract Background: Irofulven is a semisynthetic sesquiterpene anticancer agent initially investigated for its unique mechanism of action and potential to overcome resistance observed in other chemotherapeutics. This study investigates the pharmacokinetics and pharmacodynamics of a second generation irofulven analog, CAP-121 (Califia Pharma), and its efficacy and safety in preclinical cancer models relative to irofulven. Methods: 24-hour comprehensive PK was performed at two different intravenous (IV) doses in canines for irofulven (0.2 mg/kg and 1 mg/kg; MTD) and CAP-121 (1 mg/kg and 3 mg/kg). Plasma concentrations were measured by ultra-high performance liquid chromatography multiple reaction monitoring mass spectrometry. PK parameters were determined for both compounds using noncompartmental methods (Phoenix WinNonlin, version 8.4). PK parameters included area under the concentration vs time curve (AUC), maximum observed concentration (Cmax), clearance (CL), volume of distribution (Vd), and elimination half-life (t1/2). Canine platelet and neutrophil counts were measured across 28 days to ascertain the maximum tolerated dose (MTD); platelet count reversibility was also assessed. Mice xenograft models implanted with a drug resistant human ovarian cancer (A2870), or a drug-resistant human metastatic lung cancer (MV522), were dosed with irofulven (9 mg/kg; MTD) and CAP-121 (15, 20, and 25 mg/kg) and resulting tumor mass, % change in body weight, and platelet counts were compared relative to vehicle only. Results: Systemic exposure, CL, Vd, and t1/2 were similar between irofulven and CAP-121 at a 1 mg/kg IV dose. For irofulven, exposure increased approximately proportionally at doses of 0.2 mg/kg and 1 mg/kg. For CAP-121, exposure increased less than proportionally at doses of 1 mg/kg and 3 mg/kg. MTD of CAP-121 was found to be 3-fold higher in canines compared to irofulven as 3-fold more CAP-121 was required to match the platelet reduction with irofulven; platelet nadir was rapidly reversible for CAP-121. Canine nadir platelets were 3-fold higher for CAP-121 vs irofulven at the same dose (1 mg/kg). CAP-121 was superior to irofulven (p < 0.02) with regards to final tumor weights in mice xenograft models, even though MTD was not reached for CAP-121; a change in body weight and platelet loss was not observed for CAP-121 at up 25 mg/kg in mice. Conclusion: The canine PK/PD study of CAP-121 demonstrates that CAP-121 has favorable PK parameters. With regards to in vivo anticancer cytotoxic activity, the two drug-resistant xenograft models confirm the superiority of CAP-121 over irofulven. Together, these findings indicate a wider therapeutic window in humans is expected for CAP-121 compared to irofulven, emphasizing the potential anticancer utility of CAP-121 for solid cancers without prolonged thrombocytopenia. Further investigation in clinical trials is warranted Citation Format: Raymond T. Suhandynata, T K. Nguyen, Venkata R. Kotamraju, Jeremiah D. Momper, Michael J. Kelner. Safety, efficacy, pharmacokinetics, and pharmacodynamics of a second generation Irofulven analog CAP-0121 in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5925.

Profiling of Oxylipins as Markers of Oxidative Stress in Biological Samples.

Oxylipins are oxidized metabolites of polyunsaturated fatty acids (PUFAs). They represent a class of risk markers and/or therapeutic targets for diseases associated with inflammation, including cardiovascular disease and brain disorders. Because the biological activities of free PUFAs and oxylipins depend on their chemical structures and concentrations, monitoring PUFAs and oxylipin levels in biological systems is critical for understanding their roles in health and disease. Traditionally, accurate quantification of free PUFAs and oxylipins in biological samples was performed separately, as PUFAs are often 1000-fold more abundant than the derived oxidized fatty acids (oxylipins). This article describes a liquid chromatography multiple reaction monitoring tandem mass spectrometry method for the quantitative analysis of five free PUFAs and 88 oxylipins in various biological fluids, including plasma, platelet supernatants, and tissues. The same approach can also be used in conjunction with an alkaline hydrolysis step to quantify total oxylipins in fish oils. We observed that in some samples, linoleic acid levels in plasma and eicosapentaenoic acid and arachidonic acid levels in brain tissue were above the upper limit of quantification. To address this issue, we developed a data analysis method to obtain PUFA and oxylipin concentrations in these samples without additional sample preparation, thus significantly saving time and labor. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantification of polyunsaturated fatty acids (PUFAs) and oxylipins using liquid chromatography multiple reaction monitoring tandem mass spectrometry Support Protocol 1: Preparation of internal standard mixed working solution Support Protocol 2: Preparation of standard mixed stock solution Support Protocol 3: Preparation of standard mixed working solution Alternate Protocol 1: Extraction and quantitation of free PUFAs and oxylipins from mouse brain tissue Alternate Protocol 2: Extraction and quantitation of total PUFAs and oxylipins from fish oil.

Quantitative Proteomics of Maternal Blood Plasma in Isolated Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) remains a significant concern in modern obstetrics, linked to high neonatal health problems and even death, as well as childhood disability, affecting adult quality of life. The role of maternal and fetus adaptation during adverse pregnancy is still not completely understood. This study aimed to investigate the disturbance in biological processes associated with isolated IUGR via blood plasma proteomics. The levels of 125 maternal plasma proteins were quantified by liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) with corresponding stable isotope-labeled peptide standards (SIS). Thirteen potential markers of IUGR (Gelsolin, Alpha-2-macroglobulin, Apolipoprotein A-IV, Apolipoprotein B-100, Apolipoprotein(a), Adiponectin, Complement C5, Apolipoprotein D, Alpha-1B-glycoprotein, Serum albumin, Fibronectin, Glutathione peroxidase 3, Lipopolysaccharide-binding protein) were found to be inter-connected in a protein–protein network. These proteins are involved in plasma lipoprotein assembly, remodeling, and clearance; lipid metabolism, especially cholesterol and phospholipids; hemostasis, including platelet degranulation; and immune system regulation. Additionally, 18 proteins were specific to a particular type of IUGR (early or late). Distinct patterns in the coagulation and fibrinolysis systems were observed between isolated early- and late-onset IUGR. Our findings highlight the complex interplay of immune and coagulation factors in IUGR and the differences between early- and late-onset IUGR and other placenta-related conditions like PE. Understanding these mechanisms is crucial for developing targeted interventions and improving outcomes for pregnancies affected by IUGR.

Integrating Proteomics and Lipidomics for Evaluating the Risk of Breast Cancer Progression: A Pilot Study.

Metastasis is a serious and often life-threatening condition, representing the leading cause of death among women with breast cancer (BC). Although the current clinical classification of BC is well-established, the addition of minimally invasive laboratory tests based on peripheral blood biomarkers that reflect pathological changes in the body is of utmost importance. In the current study, the serum proteome and lipidome profiles for 50 BC patients with (25) and without (25) metastasis were studied. Targeted proteomic analysis for concertation measurements of 125 proteins in the serum was performed via liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) using the BAK 125 kit (MRM Proteomics Inc., Victoria, BC, Canada). Untargeted label-free lipidomic analysis was performed using liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS), in both positive and negative ion modes. Finally, 87 serum proteins and 295 lipids were quantified and showed a moderate correlation with tumor grade, histological and biological subtypes, and the number of lymph node metastases. Two highly accurate classifiers that enabled distinguishing between metastatic and non-metastatic BC were developed based on proteomic (accuracy 90%) and lipidomic (accuracy 80%) features. The best classifier (91% sensitivity, 89% specificity, AUC = 0.92) for BC metastasis diagnostics was based on logistic regression and the serum levels of 11 proteins: alpha-2-macroglobulin, coagulation factor XII, adiponectin, leucine-rich alpha-2-glycoprotein, alpha-2-HS-glycoprotein, Ig mu chain C region, apolipoprotein C-IV, carbonic anhydrase 1, apolipoprotein A-II, apolipoprotein C-II and alpha-1-acid glycoprotein 1.

The generation of 5-N-glycolylneuraminic acid as a consequence of high levels of reactive oxygen species.

The presence of N-glycolylneuraminic acid (Neu5Gc), a non-human sialic acid in cancer patients, is currently attributed to the consumption of red meat. Excess dietary red meat has been considered a risk factor causing chronic inflammation and for the development of cancers. However, it remains unknown whether Neu5Gc can be generated via a chemical reaction rather than via a metabolic pathway in the presence of high levels of reactive oxygen species (ROS) found in the inflammatory and tumor environments. In this study, the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc has been assessed in vitro under conditions mimicking the hydroxyl radical-rich humoral environment found in inflammatory and cancerous tissues. As a result, Neu5Gc has been detected via liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, this conversion has also been found to take place in serum biomatrix containing ROS and in cancer cell cultures with induced ROS production.

Deciphering Phenotypes from Protein Biomarkers for Translational Research with PIPER.

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use. LC-MRM panels provide the capability to quantify clinical biomarkers and emerging protein markers to establish the context of tumor phenotypes that provide highly relevant supporting information. An application to visualize and communicate targeted proteomics data will empower translational researchers to move protein biomarker panels from discovery to clinical use. Therefore, we have developed a web-based tool for targeted proteomics that provides pathway-level evaluations of key biological drivers (e.g., EGFR signaling), signature scores (representing phenotypes) (e.g., EMT), and the ability to quantify specific drug targets across a sample cohort. This tool represents a framework for integrating summary information, decision algorithms, and risk scores to support Physician-Interpretable Phenotypic Evaluation in R (PIPER) that can be reused or repurposed by other labs to communicate and interpret their own biomarker panels.

Effect of Xenogeneic Substances on the Glycan Profiles and Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring- mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

Targeted Proteomic Profiling Revealed Roles of Small GTPases during Osteogenic Differentiation.

The small GTPase superfamily of proteins are crucial for numerous cellular processes, including early development. The roles of these proteins in osteogenic differentiation, however, remained poorly explored. In this study, we employed a high-throughput targeted proteomic method, relying on scheduled liquid chromatography-multiple-reaction monitoring (LC-MRM) coupled with synthetic stable isotope-labeled peptides, to interrogate systematically the temporal responses of the entire small GTPase proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. Our results demonstrated that the method offers high quantification accuracy, reproducibility, and throughput. In addition, the quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved with autophagy. We also documented a previously unrecognized role of KRAS in osteogenesis, where it regulates the accumulation of extracellular matrix for mineralization through attenuating the activity of secreted matrix metalloproteinase 9 (MMP9). Together, this study represents a novel application of a state-of-the-art analytical method, i.e., targeted quantitative proteomics, for revealing the progressive reprogramming of the small GTPase proteome during osteogenic differentiation of human embryonic stem cells, and our results revealed KRAS as a new regulator for osteogenesis.

Analysis of serum reproductive hormones and ovarian genes in pubertal female goats

BackgroundAge at puberty is an important factor affecting goat fertility, with endocrine and genetic factors playing a crucial role in the onset of puberty. To better understand the relationship between endocrine and genetic factors and mechanisms underlying puberty onset in goats, reproductive hormone levels were analyzed by ELISA and ultraperformance liquid chromatography–multiple reaction monitoring–multistage/mass spectrometry and RNA sequencing was performed to analyze ovarian genes.ResultsSerum follicle stimulating hormone, luteinizing hormone, estradiol, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone, cortisone, and cortisol levels were found to be higher but progesterone were lower in pubertal goats as compared to those in prepubertal goats (P < 0.05). A total of 18,139 genes were identified in cDNA libraries, and 75 differentially expressed genes (DEGs) were identified (|log2 fold change|≥ 1, P ≤ 0.05), of which 32 were significantly up- and 43 were down-regulated in pubertal goats. Gene ontology enrichment analyses indicated that DEGs were mainly involved in “metabolic process,” “signaling,” “reproduction,” and “growth.” Further, DEGs were significantly enriched in 91 Kyoto Encyclopedia of Genes and Genomes pathways, including estrogen signaling pathway, steroid hormone biosynthesis, and cAMP signaling pathway. Bioinformatics analysis showed that PRLR and THBS1 were highly expressed in pubertal ovaries, and ZP3, ZP4, and ASTL showed low expression, suggesting their involvement in follicular development and lutealization.ConclusionsTo summarize, serum hormone changes and ovarian DEGs expression were investigated in our study. Further studies are warranted to comprehensively explore the functions of DEGs in goat puberty.

Comparison of LC-MS3 and LC-MRM Methods for Quantifying Amantadine and Its Application in Therapeutic Amantadine Monitoring in Human Plasma.

A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS3). The LC-MS3 system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass spectrometer. First, the plasma samples were pretreated using acetonitrile as the extracting solution to precipitate protein. Next, amantadine and amantadine-d15 (AMT-d15) were separated on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 50 mm, 2.7 μm) using isocratic elution with solvent A (70% 0.1% formic acid) and solvent B (30% acetonitrile) at a flow rate of 0.8 mL/min. The total run time for each sample was 3 min. The system used triple-stage fragmentation transitions at m/z 152.2→135.3→107.4 for AMT quantification in the positive ion mode and m/z 167.0→150.3→118.1 for AMT-d15 quantification. The LC-MS3 assay was linear (r &gt; 0.995) with a concentration range of 50-1500 ng/mL. The lower limit of quantification (LLOQ) was 50 ng/mL, and the intra-day and inter-day accuracies and precisions were less than 8.0% at all concentrations. In addition, the recoveries and matrix effect for AMT in human plasma were within acceptable limits. In terms of stability, AMT had no significant degradation under all conditions. All the results met the requirements of the guidelines of the Food and Drug Administration (FDA) for biological method validation. The novelty of the MS3 assay was that it presented a methodology with higher selectivity and sensitivity. This method was successfully applied to 44 human plasma samples, and the obtained quantitative results were compared with another liquid chromatography-multiple reaction monitoring (LC-MRM) method. The Passing-Bablok regression coefficients and Bland-Altman plot revealed no difference between the LC-MS3 and LC-MRM methods, implying that the developed LC-MS3 method is a reliable and accurate assay for AMT determination in human plasma. These results are also a proof of concept for determining chemicals in biological samples by the LC-MS3 strategy.

Direct acetylation for full analysis of polysaccharides in edible plants and fungi using reverse phase liquid chromatography-multiple reaction monitoring mass spectrometry

It is vitally important to characterize polysaccharides by monosaccharide composition method. In this study, a direct acetylation strategy combined with reversed-phase liquid chromatography electrospray tandem multiple reaction monitoring mass spectrometry (RPLC-ESI-MRM-MS) was developed for simultaneous determination of 8 aldoses (Glc, Gal, Man, Ara, Xyl, Rib, Rha and Fuc), a ketose (Fru), 2 alditols (Glc-ol and Man-ol) and 2 uronic acids (GlcA and GalA) on a high-pressure resistant reversed-phase column. Employing 1-MeIm as catalyst for direct acetylation, even though no DMSO was used to inhibit the transformation of configurations, each carbohydrate still produced a single chromatographic peak in RPLC conditions due to the ɑ- and β- isomers merged together. Except for Fru and Man, all the other 11 carbohydrates were base-line separated in a 1.7 µm CYANO column. Therefore, correction factor method is further proposed to perfectly solve co-elution problem of Fru and Man because of occurrence of a specific Q3 ion for aldoses rather than ketose. The result was verified on a 1.7 µm Fluoro-Phenyl column with a full separation of Fru and Man. Herein, the established direct acetylation as followed RPLC-ESI-MRM-MS method was successfully applied for compositional analysis of complex polysaccharides from edible plants and fungi.

Extraction and Simultaneous Quantification of Endocannabinoids and Endocannabinoid-Like Lipids in Biological Tissues.

Extraction and quantification of endocannabinoids from biological tissues is essential to unravel their changes under physiological and pathophysiological conditions. We describe here an analytical protocol for the extraction of endocannabinoids, anandamide (archidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and endocannabinoid-like lipids such as palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA), as well as arachidonic acid (AA) from biological tissues using liquid-liquid extraction method and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM).

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Accelerating the Validation of Endogenous On-Target Engagement and In Cellulo Kinetic Assessment for Covalent Inhibitors of KRASG12C in Early Drug Discovery.

Covalent inhibition is a valuable modality in drug discovery because of its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in vitro inhibition assessment to the appropriate in cellulo target engagement parameter by covalent probes. Estimation of such parameters is often impeded by the low-throughput nature of current probe-free approaches. In this study, an ultra-performance liquid chromatography-multiple reaction monitoring mass spectrometry platform was utilized to develop a targeted proteomics workflow that can evaluate cellular on-target engagement of covalent molecules in an increased throughput manner. This workflow enabled a throughput increase of 5-10 fold when compared to traditional nanoLC-based proteomics studies. To demonstrate the applicability of the method, KRASG12C was used as a model system to investigate the interaction of an irreversible covalent small molecule, compound 25, both in vitro and in cellulo. Initial biochemical studies confirmed that the small molecule forms an adduct with the targeted cysteine on the protein, as assessed at the level of both intact protein and on the target peptide. In cellulo studies were carried out to quantify target engagement and allele selectivity assessment for the small molecule in the heterozygous NCI-H358 cell line for KRASG12C with respect to the WT type protein. The workflow enabled evaluation of in vitro and in cellulo target engagement kinetics, providing mechanistic insights into the irreversible mode of inhibition. In summary, the method has the potential for target agnostic application in the assessment of on-target engagement of covalent probes compatible with the high-throughput requirements of early drug discovery.

Synergistic effect of thermal dehydrating on the emerging contaminants removal via Electro-Fenton

Advanced oxidation processes can remove emerging contaminants from wastewater with complete degradation; however, the slow kinetics of the degradation process and extensive energy input hinder further commercialization. We developed a hybrid process, comprising electro-Fenton and membrane distillation, to simultaneously degrade organic pollutants and extract pure water from the mixture. Methyl orange, as a synthetic dye, and ibuprofen, as an emerging pharmaceutical contaminant, were used to study the effectiveness of the electro-Fenton/membrane distillation hybrid system. The dye degradation studies were conducted in three modes: electro-Fenton at room temperature, electro-Fenton at high temperatures, and electro-Fenton coupled with membrane distillation. The hybrid process showed 99% dye removal and 92% total organic carbon decay of an initial 20 ppm methyl orange after 6 h of operation. The mineralization current efficiency at room temperature was approximately 50%, which was increased by 5 and 10 times for high-temperature electro-Fenton and hybrid electro-Fenton/membrane distillation, respectively. The hybrid process also reduced the energy consumption based on the specific energy consumption results. The liquid chromatography-multiple reaction monitoring was used for quantitative profiling of ibuprofen degradation with a concentration of 0.5 ppm. The electro-Fenton/membrane distillation (20–60) removed 94% of ibuprofen after 3 h compared to the 74.7% removal rate by electro-Fenton (60).

Cerebrospinal Fluid Amino Acid Metabolite Signatures of Diabetic Cognitive Dysfunction Based on Targeted Mass Spectrometry.

Diabetic cognitive dysfunction (DCD) is one of severe diabetic complications and might develop to irreversible dementia. Early diagnosis and detection of DCD is significant for prevention and treatment. The main objective of this study was to investigate the amino acid profiles of rat with DCD in the cerebrospinal fluid (CSF) to distinguish the early specific biomarkers. In total, rats were assigned into control and model groups. Model was induced by intraperitoneal injection of streptozotocin. The Morris water maze (MWM) method was used to evaluate learning and memory in rats on the 13th week after the model established. CSF samples were collected via cisterna magna puncture at the 0th, 5th, 9th, and 13th week, and amino acids profiling of CSF samples were performed via ultra performance liquid chromatography multiple reaction monitoring mass spectrometry (UPLC-MRM-MS). The amino acid profile was processed through multivariate analysis to identify potential biomarkers, and the related metabolic pathways were analyzed by MetaboAnalyst 5.0. Compared to the control group, the escape latency of the MWM was significantly prolonged in model group rats (p < 0.05). Different amino acid profiles were obtained between two groups. L-Alanine, L-Glutamine, L-Lysine, L-Serine, and L-Threonine were identified as potential biomarkers for DCD. These biomarkers are principally involved in glycine, serine, and threonine metabolism, aminoacyl-tRNA biosynthesis, alanine, aspartate, and glutamate metabolism, and glyoxylate and dicarboxylate metabolism. There are amino acid biomarkers in the CSF of rat with DCD. The mechanism of DCD is related to those pathways, which provide help for the early diagnosis and treatment and mechanism research.

TRACES: A Lightweight Browser for Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Chromatograms.

In targeted metabolomic analysis using liquid chromatography–multiple reaction monitoring–mass spectrometry (LC-MRM-MS), hundreds of MRMs are performed in a single run, yielding a large dataset containing thousands of chromatographic peaks. Automation tools for processing large MRM datasets have been reported, but a visual review of chromatograms is still critical, as real samples with biological matrices often cause complex chromatographic patterns owing to non-specific, insufficiently separated, isomeric, and isotopic components. Herein, we report the development of new software, TRACES, a lightweight chromatogram browser for MRM-based targeted LC-MS analysis. TRACES provides rapid access to all MRM chromatograms in a dataset, allowing users to start ad hoc data browsing without preparations such as loading compound libraries. As a special function of the software, we implemented a chromatogram-level deisotoping function that facilitates the identification of regions potentially affected by isotopic signals. Using MRM libraries containing precursor and product formulae, the algorithm reveals all possible isotopic interferences in the dataset and generates deisotoped chromatograms. To validate the deisotoping function in real applications, we analyzed mouse tissue phospholipids in which isotopic interference by molecules with different fatty-acyl unsaturation levels is known. TRACES successfully removed isotopic signals within the MRM chromatograms, helping users avoid inappropriate regions for integration.

Lipidomics and Transcriptomics in Neurological Diseases.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease. Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

Prodynorphin and Proenkephalin in Cerebrospinal Fluid of Sporadic Creutzfeldt-Jakob Disease.

Proenkephalin (PENK) and prodynorphin (PDYN) are endogenous opioid peptides mainly produced in the striatum and, to a lesser extent, in the cerebral cortex. Dysregulated metabolism and altered cerebrospinal fluid (CSF) levels of PENK and PDYN have been described in several neurodegenerative diseases. However, no study to date investigated these peptides in the CSF of sporadic Creutzfeldt–Jakob disease (sCJD). Using liquid chromatography-multiple reaction monitoring mass spectrometry, we evaluated the CSF PDYN- and PENK-derived peptide levels in 25 controls and 63 patients with sCJD belonging to the most prevalent molecular subtypes (MM(V)1, VV2 and MV2K). One of the PENK-derived peptides was significantly decreased in each sCJD subtype compared to the controls without a difference among subtypes. Conversely, PDYN-derived peptides were selectively decreased in the CSF of sCJD MV2K, a subtype with a more widespread overall pathology compared to the sCJD MM(V)1 and the VV2 subtypes, which we confirmed by semiquantitative analysis of cortical and striatal neuronal loss and astrocytosis. In sCJD CSF PENK and PDYN were associated with CSF biomarkers of neurodegeneration but not with clinical variables and showed a poor diagnostic performance. CSF PDYN and PENK-derived peptides had no significant diagnostic and prognostic values in sCJD; however, the distinct marker levels between molecular subtypes might help to better understand the basis of phenotypic heterogeneity determined by divergent neuronal targeting.