Mass Spectrometry-based Methods Research Articles

#983 Identification of gut microbiome signatures associated with albuminuria in diabetes mellitus

Abstract Background and Aims Diabetes is a growing global problem with serious complications, including kidney impairment and cardiovascular morbidity and mortality. Monitoring albuminuria, which is associated with these complications, is crucial in optimal diabetes management. Recent research suggests a link between the gut microbiome, plasma metabolites and albuminuria development in diabetes patients, highlighting the need for further investigation using deep metagenomic sequencing. Method We investigated the relationship between 1002 gut microbial species, 1308 metabolites and albuminuria in 752 participants with diabetes from the Swedish CArdioPulmonary BioImage Study. To determine the relative abundance of species, we employed deep shotgun metagenomic sequencing of fecal samples. Plasma metabolites were analyzed using mass spectrometry-based methods. Results Shannon diversity index was inversely associated with albuminuria after adjusting for confounding factors (Fig. 1). Our multivariable-adjusted models revealed seven metagenomic species that were associated with albuminuria (Fig. 2). Furthermore, employing a global metabolomics approach, we identified phenyl sulfate, among 98 annotated metabolites (Fig. 3), as linked to the species Clostridium sp. AT4 and Eggerthella lenta. Notably, phenyl sulfate is involved in tyrosine metabolism, and we also observed an association between the gut metabolic module of Tyrosine degradation II and albuminuria. Conclusion In the present study, we identified seven gut microbial species linked to albuminuria and established a connection between these species and phenyl sulfate, which previously has been associated with podocyte damage and the development of albuminuria. These findings provided additional evidence of the impact of microbial species and contribute to our understanding of the complex relationship between the gut microbiome, plasma metabolites, and albuminuria in patients with diabetes.

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress.

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis (Arabidopsis thaliana) leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant trigalactosyldiacylglycerol1-1 sugar dependent 1-4 (tgd1-1 sdp1-4) that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we showed that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress.

Rapid and Sensitive Detection of Fentanyl and Its Analogs by a Novel Chemiluminescence Immunoassay.

Abuse of fentanyl and its analogs is a major contributor to the opioid overdose epidemic in the United States, but detecting and quantifying trace amounts of such drugs remains a challenge without resorting to sophisticated mass spectrometry-based methods. A sensitive immunoassay with a sub-picogram limit of detection for fentanyl and a wide range of fentanyl analogs has been developed, using a novel high-affinity antibody fused with NanoLuc, a small-size luciferase that can emit strong and stable luminescence. When used with human urine samples, the assay has a sub-picogram limit of detection for fentanyl, with results fully concordant with LC-MS. When applied to clinical samples, the novel chemiluminescence immunoassay can detect low positive fentanyl missed by routine screening immunoassays, with a limit of detection of 0.8 pg/mL in human urine. When applied to environmental samples, the assay can detect levels as low as 0.25 pg fentanyl per inch2 of environment surface. Assay turnaround time is less than 1 h, with inexpensive equipment and the potential for high-throughput automation or in-field screening. We have established a novel assay that may have broad applications in clinical, environmental, occupational, and forensic scenarios for detection of trace amounts of fentanyl and its analogs.

SLIM-Based High-Resolution Ion Mobility Reveals New Structural Insights into Isomeric Vitamin D Metabolites and their Isotopologues.

Testing for vitamin D deficiency remains a high-volume clinical assay, much of which is done using mass spectrometry-based methods to alleviate challenges in selectivity associated with immunoassays. Ion mobility-mass spectrometry (IM-MS) has been proposed as a rapid alternative to traditional LC-MS/MS methods, but understanding the structural ensemble that contributes to the ion mobility behavior of this molecular class is critical. Herein we demonstrate the first application of high-resolution Structures for Lossless Ion Manipulations (SLIM) IM separations of several groups of isomeric vitamin D metabolites. Despite previous IM studies of these molecules, the high resolving power of SLIM (Rp ∼ 200) has revealed additional conformations for several of the compounds. The highly similar collision cross sections (CCS), some differing by as little as 0.7%, precluded adequate characterization with low-resolution IM techniques where, in some cases, wider than expected peak widths and/or subtle shoulders may have hinted at their presence. Importantly, these newly resolved peaks often provided a unique mobility that could be used to separate isomers and provides potential for their use in quantification. Lastly, the contribution of isotopic labeling to arrival time distribution for commonly used 13C- and deuterium-labeled internal standards was explored. Minor shifts of ∼0.2-0.3% were observed, and in some instances these shifts were specific to the conformer being measured (i.e., "closed" vs "open"). Accounting for these shifts is important during raw data extraction to ensure reproducible peak area integration, which will be a critical consideration in future quantitative applications.

Coupling imaging mass cytometry with Alcian blue histochemical staining for a single-slide approach.

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.

Key Proteomics Tools for Fundamental and Applied Microalgal Research.

Microscopic, photosynthetic prokaryotes and eukaryotes, collectively referred to as microalgae, are widely studied to improve our understanding of key metabolic pathways (e.g., photosynthesis) and for the development of biotechnological applications. Omics technologies, which are now common tools in biological research, have been shown to be critical in microalgal research. In the past decade, significant technological advancements have allowed omics technologies to become more affordable and efficient, with huge datasets being generated. In particular, where studies focused on a single or few proteins decades ago, it is now possible to study the whole proteome of a microalgae. The development of mass spectrometry-based methods has provided this leap forward with the high-throughput identification and quantification of proteins. This review specifically provides an overview of the use of proteomics in fundamental (e.g., photosynthesis) and applied (e.g., lipid production for biofuel) microalgal research, and presents future research directions in this field.

Insight into sodium selenite-induced metabolic reprogramming in response to main seleno-amino acid enrichment in Saccharomyces cerevisiae using metabolomics

Conversion of inorganic selenium (Se) to its organic form by Saccharomyces cerevisiae (S. cerevisiae) has attracted significant attention, for organic Se has higher nutritional value with less negative effects. However, the mechanism of transformation and accumulation of organic Se at the metabolic level remains unclear. We investigated the metabolic response to sodium selenite supplementation in culture medium at 0, 5, 15, and 45 mg/L in two S. cerevisiae strains using an ultra-high performance liquid chromatography-high resolution mass spectrometry-based untargeted method. In total, 455 metabolites were detected, and a separation trend was clearly observed between Se-treated groups and controls. In depth, metabolic pathways involving amino acid, nucleotide metabolism and tricarboxylic acid cycle were significantly affected. Furthermore, high inorganic-Se supplementation effectively decreased the seleno-amino acid proportion in total Se, inhibited cell growth, and decreased glutathione levels. These results provide novel insights into the Se-enrichment process of S. cerevisiae.

Abstract 5376: Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow

Abstract Major histocompatibility complex (MHC) molecules play a central role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. In the context of cancer, understanding the repertoire of tumor-associated antigens (TAAs) presented by MHC molecules (or HLA molecules in human) is crucial for deciphering how the immune system recognizes and responds to malignant cells. The identification of neoantigens, unique to individual tumors due to somatic mutations, has become a focal point in immunopeptidomic studies. One significant hurdle in systematic immunopeptidomics analysis is the high input material requirement. Here, we present a semi-automated workflow to robustly identify and quantify immunopeptides from reduced amounts of clinical tissue biopsy and peripheral blood mononuclear cell samples. At the core of immunopeptidomics is the enrichment of HLA-associated peptides, followed by identification using mass spectrometry and bioinformatics tools. We optimized the native lysis and a sequential immunoprecipitation workflow for both class I and class II immunopeptides while ensuring scalability and reproducibility. Leveraging the magnetic properties of the beads, 1,000 samples can be processed within a week by a single operator. For both tissue and PBMC samples, we performed a systematic ramping experiment starting with as little as 2.5mg tissue or 5 million PBMCs. In all experiments, The established sample preparation offers high reproducibility and identifications of good quality: 1) Class-I immunopeptides: >60% of the peptides identified are 9-mers, >80% predicted strong binders, and the expected amino acids are enriched at the anchor positions; 2) Class-II immunopeptides, >50% of the peptides identified are 14-to-16-mers, and >50% are predicted strong binders. Furthermore, the pipeline is highly sensitive as we could still identify over 2,800 class-I immunopeptides when processing 2.5 mg fresh frozen tissue and 2,000 - 3,000 class-I immunopeptides when starting from 5 million PBMCs. Furthermore, the developed immunopeptidomics workflow was deployed to profile a cohort of 12 cancerous and matched healthy lung tissue samples. Their Class-I immunopeptidomes clearly displayed a pattern where matched tissues from each subject cluster together, further underlining the fact that the intricate immune-tumor interface is highly personalized. Overall, we established a robust pipeline for for class-I and II immunopeptidome profiling from clinically relevant sample types. Taking advantage of the nature of mass spectrometry-based methods, customized targeted assays can be developed without the need for affinity reagent, allowing specific and absolute quantification of any immunopeptide of interest. Citation Format: Ilja E. Shapiro, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Daniel Redfern, Yuehan Feng, Roland Bruderer, Lukas Reiter. Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow [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 5376.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 μg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups.This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis.Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD.The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.

Development of a Mass Spectrometry-Based Method for Quantification of Total Cashew Protein in Roasting Oil.

Food allergen cross-contact during food preparation and production is one of the causes of unintentional allergen presence in packaged foods. However, little is known about allergen cross-contact in shared frying or roasting oil, which prevents the establishment of effective allergen controls and may put allergic individuals at risk. To better understand the quantity of allergen transferred to frying oil and subsequent products, an analytical method is needed for quantifying protein in oil that has been exposed to frying/roasting conditions. The goal of this study was to develop a parallel reaction monitoring LC-MS/MS method to quantify the amount of cashew protein in shared roasting oil. The sample preparation method was evaluated to improve protein extractability and peptide performance. Four quantitative peptides representing cashew 2S and 11S proteins were selected as targets based on their sensitivity, heat stability, and specificity. A calibration strategy was developed to quantify the amount of total cashew protein in oil. Method performance was evaluated using a heated cashew-in-oil model system. The method showed high recovery in oil samples spiked with 100 or 10 parts per million (ppm) total cashew protein heated at 138 or 166°C for 2-30 min. Samples (100 ppm total cashew protein) heated for 30 min had more than 90% recovery when treated at 138°C and more than 50% when heated at 166°C. The method is fit-for-purpose for the analysis of cashew allergen cross-contact in oil. A novel MS-based method was developed that can accurately quantify the amount of cashew protein present in heated oil.

An LC-MS/MS-based method for the simultaneous quantification of melatonin, cortisol and cortisone in saliva

Disturbances in the diurnal pattern are associated with several clinical and psychological conditions, including depression and fatigue. Salivary sampling for melatonin, cortisol and cortisone provides a non-invasive method for frequent sampling and obtaining biochemical insight into the diurnal pattern of individuals. Therefore, a new liquid chromatography-tandem mass spectrometry-based method for the measurement of salivary melatonin, cortisol and cortisone was developed and validated. The method required 250 μl saliva, used isotope dilution methodology and was based on a liquid-liquid extraction for sample preparation, reversed-phase chromatography and multiple reaction monitoring on a mass spectrometer for quantitation. The lower limits of quantification obtained were 0.010 nmol/L for melatonin, 0.5 nmol/L for cortisol and 1.00 nmol/L for cortisone and the limits of detection were 0.003 nmol/L, 0.15 nmol/L and 0.1 nmol/L respectively. The method imprecision was ≤14% for all measurands, and the method comparison showed highly comparable results with high correlation coefficients (all ≥0.964). Potential interference of cortisol and cortisone by prednisolone was observed and could be detected by chromatogram review. Typical diurnal patterns for melatonin, cortisol and cortisone were observed in the saliva of 20 cancer survivors who collected saliva throughout the day.

Measurement of CYP1A2 and CYP3A4 activity by a simplified Geneva cocktail approach in a cohort of free-living individuals: a pilot study.

Introduction: The cytochrome P450 enzyme subfamilies, including CYP3A4 and CYP1A2, have a major role in metabolism of a range of drugs including several anti-cancer treatments. Many factors including environmental exposures, diet, diseaserelated systemic inflammation and certain genetic polymorphisms can impact the activity level of these enzymes. As a result, the net activity of each enzyme subfamily can vary widely between individuals and in the same individual over time. This variability has potential major implications for treatment efficacy and risk of drug toxicity, but currently no assays are available for routine use to guide clinical decision-making. Methods: To address this, a mass spectrometry-based method to measure activities of CYP3A4, CYP1A2 was adapted and tested in free-living participants. The assay results were compared with the predicted activity of each enzyme, based on a self-report tool capturing diet, medication, chronic disease state, and tobacco usage. In addition, a feasibility test was performed using a low-volume dried blood spots (DBS) on two different filter-paper supports, to determine if the same assay could be deployed without the need for repeated standard blood tests. Results: The results confirmed the methodology is safe and feasible to perform in free-living participants using midazolam and caffeine as test substrates for CYP3A4 and CYP1A2 respectively. Furthermore, though similar methods were previously shown to be compatible with the DBS format, the assay can also be performed successfully while incorporating glucuronidase treatment into the DBS approach. The measured CYP3A4 activity score varied 2.6-fold across participants and correlated with predicted activity score obtained with the self-report tool. The measured CYP1A2 activity varied 3.5-fold between participants but no correlation with predicted activity from the self-report tool was found. Discussion: The results confirm the wide variation in CYP activity between individuals and the important role of diet and other exposures in determining CYP3A4 activity. This methodology shows great potential and future cross-sectional and longitudinal studies using DBS are warranted to determine how best to use the assay results to guide drug treatments.

Longitudinal analysis of external quality assessment of immunoassay-based steroid hormone measurement indicates potential for improvement in standardization.

As hormonal disorders are linked to several diseases, the accurate quantitation of steroid hormone levels in serum is crucial in order to provide patients with a reliable diagnosis. Mass spectrometry-based methods are regarded as having the highest level of specificity and sensitivity. However, immunoassays are more commonly used in routine diagnostics to measure steroid levels as they are more cost effective and straightforward to conduct. This study analyzes the external quality assessment results for the measurement of testosterone, progesterone and 17β-estradiol in serum using immunoassays between early 2020 and May 2022. As reference measurement procedures are available for the three steroid hormones, the manufacturer-specific biases were normalized to the reference measurement values. The manufacturer-specific coefficients of variation were predominantly inconspicuous, below 20% for the three hormones when outliers are disregarded, however there were large differences between the various manufacturer collectives. For some collectives, the median bias to the respective reference measurement value was repeatedly greater than ±35%, which is the acceptance limit defined by the German Medical Association. In the case of testosterone and progesterone determination, some collectives tended to consistently over- or underestimate analyte concentrations compared to the reference measurement value, however, for 17β-estradiol determination, both positive and negative biases were observed. This insufficient level of accuracy suggests that cross-reactivity continues to be a fundamental challenge when antibody detection is used to quantify steroids with a high structural similarity. Distinct improvements in standardization are required to provide accurate analysis and thus, reliable clinical interpretations. The increased accuracy of the AX immunoassay for testosterone measurement, as observed in the INSTAND EQAs between 2020 and 2022, could be the result of a recalibration of the assay and raises hope for further improvement of standardization of immunoassay-based steroid hormone analyses in the coming years.

Interaction network of human early embryonic transcription factors

Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.

Systematic analysis of nonprogrammed frameshift suppression in E. coli via translational tiling proteomics

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the -1 frame, occur at specific evolutionarily optimized "slippery" sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry-based method called "translational tiling proteomics" for interrogating the full set of possible -1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical "slippery" sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed.

Development and validation of tandem mass spectrometry-based method for the analysis of more than 400 pesticides in honey

A highly sensitive, efficient and rugged analytical method was developed and validated for determination of residues of more than 400 multiclass pesticides in honey using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach in combination with liquid and gas chromatography coupled to tandem mass spectrometry (LC-MS/MS and GC-MS/MS). Various combinations of extracting solvents and cleanup sorbents were evaluated to optimize the sample preparation step. The method involving acetonitrile as extracting solvent and primary secondary amine (PSA) with magnesium sulphate (MgSO4) as cleanup sorbent proved to be the most effective. The analytical method was validated at 5, 10, and 20 µg kg−1 in accordance with SANTE/11312/2021 and FDA guidelines and found suitable for simultaneous determination of 206 pesticides on LC-MS/MS and 197 pesticides on GC-MS/MS with acceptable recovery (70–120%), precision (RSD <20.0%), linearity (correlation coefficient ≥ 0.99) at limit of quantification (LOQ) ranging from 5 and 10 µg kg−1. The method was successfully applied to monitor pesticide residues in 80 market honey samples collected from different parts of India. The developed method enabled the quantitative determination of a wide spectrum of pesticide residues in honey in a single chromatographic run.

Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins.

Drug resistance remains one of the main causes of poor outcome in cancer therapy. It is also becoming evident that drug resistance to both chemotherapy and to antibiotics is driven by more than one mechanism. So far, there are at least eight recognized mechanisms behind such resistance. In this review, we choose to discuss one of these mechanisms, which is known to be partially driven by a class of transmembrane proteins known as ATP-binding cassette (ABC) transporters. In normal tissues, ABC transporters protect the cells from the toxic effects of xenobiotics, whereas in tumor cells, they reduce the intracellular concentrations of anticancer drugs, which ultimately leads to the emergence of multidrug resistance (MDR). A deeper understanding of the structures and the biology of these proteins is central to current efforts to circumvent resistance to both chemotherapy, targeted therapy, and antibiotics. Understanding the biology and the function of these proteins requires detailed structural and conformational information for this class of membrane proteins. For many years, such structural information has been mainly provided by X-ray crystallography and cryo-electron microscopy. More recently, mass spectrometry-based methods assumed an important role in the area of structural and conformational characterization of this class of proteins. The contribution of this technique to structural biology has been enhanced by its combination with liquid chromatography and ion mobility, as well as more refined labelling protocols and the use of more efficient fragmentation methods, which allow the detection and localization of labile post-translational modifications. In this review, we discuss the contribution of mass spectrometry to efforts to characterize some members of the ATP-binding cassette (ABC) proteins and why such a contribution is relevant to efforts to clarify the link between the overexpression of these proteins and the most widespread mechanism of chemoresistance.

Metabolomics: a review of liquid chromatography mass spectrometry-based methods and clinical applications

Abstract Introduction Metabolomics is a rapidly growing field that aims to understand the complex metabolic pathways involved in health and disease. Liquid chromatography mass spectrometry (LC-MS) based untargeted metabolomics has emerged as a powerful tool for investigating the metabolic changes associated with various diseases, and for identifying potential biomarkers for early disease detection and treatment monitoring. This review provides a comprehensive overview of LC-MS based untargeted metabolomics and its clinical applications. Content The advantages and challenges of untargeted metabolomics are discussed, encompassing sample preparation, data processing, sample storage, acquisition mode selection, column strategy, and annotation. The latest advancements in LC-MS technology and data analysis techniques are reviewed, which have facilitated the more accurate and sensitive detection of metabolites in biological samples. The clinical applications of untargeted metabolomics are emphasized, including its utilization in disease diagnosis, treatment monitoring, and personalized medicine. Examples are provided of how biomarkers for various diseases, such as cancer, coronary heart disease, and infectious disease, have been identified through the application of untargeted metabolomics. Summary and outlook Overall, LC-MS based untargeted metabolomics is a powerful tool for investigating the complex metabolic changes associated with various diseases, and has the potential to transform clinical diagnosis, treatment, and personalized medicine.

Pharmacokinetics, absolute bioavailability, and tissue distribution of WJ-14, a novel N-methyl-d-aspartate receptor antagonist, in rats by liquid chromatography-tandem mass spectrometry.

To circumvent the limitations of current antidepressants, WJ-14, a novel N-methyl-d-aspartate receptor antagonist, was synthesized and demonstrated to have remarkable efficiency in the treatment of depression. To illustrate the pharmacokinetics, absolute bioavailability, and tissue distribution of WJ-14 in rats, a rapid and sensitive liquid chromatography-tandem mass spectrometry-based analytical method was developed and validated for the separation and detection of WJ-14 in both plasma and tissue samples. After oral administration, WJ-14 was rapidly absorbed into the blood with time to reach the maximum plasma concentration (Tmax ) within 0.28 h and quickly eliminated with clearance (Cl) exceeding 6.80L/h/kg and elimination half-life (t1/2 ) within 2.69 h. No obvious accumulation was found with mean residencetime (MRT) within 4.10 h. Tissue distribution revealed that WJ-14 was extensively distributed in the main tissues of rats, and massive amounts of WJ-14 were distributed in the liver. Extensive distribution and quick elimination led to extremely low absolute bioavailability of WJ-14 (1.91% of 8.33 mg/kg and 3.30% of 24.99 mg/kg). WJ-14 was detected in the brain only 0.083 h after oral administration, which is crucial for a rapid-onset antidepressant candidate. In addition, WJ-14 likely exhibited a non-linear pharmacokinetic process at dosages of 8.33 and 24.99 mg/kg. The findings may provide valuable information for subsequent studies on WJ-14.