Quantification Of Small Molecules Research Articles

Metabolomic Changes in Plasma of Preterminal Stage of Rhesus Nonhuman Primates Exposed to Lethal Dose of Radiation.

Ionizing radiation exposure is known to induce molecular and cellular injury, inflicting a cascade of potentially catastrophic events leading to tissue and organ damage. Metabolomic analysis allows for the identification and quantification of small molecules downstream of genomic changes induced by radiation exposure. We aimed to characterize metabolomic changes that underscore the prefinal stage of lethally irradiated rhesus nonhuman primates (NHPs). Peripheral blood was drawn at baseline, post-exposure, as well as at the preterminal stage in NHPs (immediately prior to death in moribund NHPs) that did not survive exposure with 7.2 Gy total-body radiation (LD70/60). Herein, we analyzed global metabolomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in plasma samples of NHPs collected at various timepoints in relation to irradiation. The overall goal was to identify metabolic shifts present immediately prior to death. Our findings showed radiation induced significant time-dependent metabolic perturbations when compared to pre-irradiation profiles, particularly in glycerophospholipid metabolism and steroid hormone biosynthesis and metabolism pathways. These findings provide valuable insights for identifying biomarkers for lethality, which may be helpful for triage during a mass casualty scenario.

Optimizing Graphene Dopants for Direct Electrocatalytic Quantification of Small Molecules and Ions

This review critically evaluates the recent advancements in graphene dopants for electrocatalytic quantification of small molecules and ions. Emphasizing the enhanced catalytic activity and specificity of doped graphene, the paper delves into the various doping methods, ranging from chemical to physical techniques. It presents a detailed analysis of the mechanisms underlying graphene-based electrocatalysis and its applications in environmental monitoring, health care, and pharmaceuticals. The review also addresses challenges such as the reproducibility and stability of doped graphene, suggesting future research directions. By summarizing the latest findings, this review aims to elucidate the role of doped graphene in improving the sensitivity and selectivity of electrocatalytic processes, bridging the gap between research and practical use.

Ready-to-use nanopore platform for label-free small molecule quantification: Ethanolamine as first example

In recent decades, nanopores have become a promising diagnostic tool. Protein and solid-state nanopores are increasingly used for both RNA/DNA sequencing and small molecule detection. The latter is of great importance, as their detection is difficult or expensive using available methods such as HPLC or LC-MS. DNA aptamers are an excellent detection element for sensitive and specific detection of small molecules. Herein, a method for quantifying small molecules using a ready-to-use sequencing platform is described.Taking ethanolamine as an example, a strand displacement assay is developed in which the target-binding aptamer is displaced from the surface of magnetic particles by ethanolamine. Non-displaced aptamer and thus the ethanolamine concentration are detected by the nanopore system and can be quantified in the micromolar range using our in-house developed analysis software. This method is thus the first to describe a label-free approach for the detection of small molecules in a protein nanopore system.

Rapid Quantification of Alcohol Content in Intact Bottles of Wine Using Scalar-Coupled Spectroscopy at Low Field

Direct spectroscopic quantification of small molecules using low cost, low field (< 0.1 T) large bore portable magnets is not possible using conventional techniques due the presence of strong homonuclear coupling which results in complicated spectral patterns with resonances separated by much less than the achievable spectral linewidth. In contrast, a method using the signals from a Carr–Purcell–Meiboom–Gill (CPMG) train, in which the data are Fourier transformed in this indirect dimension, can produce so-called J-spectra in which several distinct spectral features can be distinguished. In this work, we evaluate this technique to quantify the amount of alcohol (ethanol) in intact bottles of wines or spirits.

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate.

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.

Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry.

Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotation (EISA) combined with correlated ion monitoring. Here we provide a step-by-step protocol for optimizing the analytical conditions for EISA, so anyone properly trained in liquid chromatography-mass spectrometry can follow and apply this technique for any given analyte. We exemplify the approach by using 2-hydroxyglutarate (2-HG) which is a clinically relevant metabolite whose D-enantiomer is considered an 'oncometabolite', characteristic of cancers associated with mutated isocitrate dehydrogenases 1 or 2 (IDH1/2). We include procedures for determining quantitative robustness, and show results of these relating to the analysis of DL-2-hydroxyglutarate in cells, as well as in serum samples from patients with acute myeloid leukemia that contain the IDH1/2 mutation. This EISA-mass spectrometry protocol is a broadly applicable and low-cost approach for the quantification of small molecules that has been developed to work well for both single-quadrupole and time-of-flight mass analyzers.

Monitoring hormone and small molecule secretion dynamics from islets-on-chip.

Tissue functions such as hormone secretion involve the interplay of multiple chemical signals and metabolic processes over time. Measuring the different components involved is useful in unraveling the interactions, but often requires use of multiple analytical techniques. The challenge of measuring the necessary components with temporal resolution is greater when tissue samples are limited. Here, an accessible microfluidic platform compatible with multiple measurement techniques to monitor cell secretions has been developed. The platform is applied to islets of Langerhans, micro-organs involved in glucose homeostasis and diabetes. The device houses 1 to 8 islets and the perfusion fluid can be controlled to change conditions, e.g., glucose concentration, in seconds. Samples are collected in fractions and split for offline analysis. The device is paired with a scaled-down immunoassay, AlphaLISA, for hormone quantification and liquid chromatography-mass spectrometry for small molecule quantification to study secretion dynamics. The combined system allows the first simultaneous measurement of insulin, glucagon, biogenic amines, and amino acids from islet secretions. The combined measurements revealed correlation in secretion events and differences in timing of release between hormones and biogenic amines and amino acids. These efforts decreased the number of islets required compared to standard approaches, thus decreasing necessary animal use, reagent use, and cost, while increasing information content achievable from one sample. The microfluidic device is a suitable platform for in-depth characterization of secretion from small tissue samples.

Plasmonically-enhanced competitive assay for ultrasensitive and multiplexed detection of small molecules

Novel methods that enable facile, ultrasensitive and multiplexed detection of low molecular weight organic compounds such as metabolites, drugs, additives, and organic pollutants are valuable in biomedical research, clinical diagnosis, food safety and environmental monitoring. Here, we demonstrate a simple, rapid, and ultrasensitive method for detection and quantification of small molecules by implementing a competitive immunoassay with an ultrabright fluorescent nanolabel, plasmonic fluor. Plasmonic-fluor is comprised of a polymer-coated gold nanorod and bovine serum albumin conjugated with molecular fluorophores and biotin. The synthesis steps and fluorescence emission of plasmonic-fluor was characterized by UV–vis spectroscopy, transmission electron microscopy, and fluorescence microscopy. Plasmon-enhanced competitive assay can be completed within 20 min and exhibited more than 30-fold lower limit-of-detection for cortisol compared to conventional competitive ELISA. The plasmon-enhanced competitive immunoassay when implemented as partition-free digital assay enabled further improvement in sensitivity. Further, spatially multiplexed plasmon-enhanced competitive assay enabled the simultaneous detection of two analytes (cortisol and fluorescein). This simple, rapid, and ultrasensitive method can be broadly employed for multiplexed detection of various small molecules in research, in-field and clinical settings.

1H-NMR metabolomics-based surrogates to impute common clinical risk factors and endpoints.

SummaryBackgroundMissing or incomplete phenotypic information can severely deteriorate the statistical power in epidemiological studies. High-throughput quantification of small-molecules in bio-samples, i.e. ‘metabolomics’, is steadily gaining popularity, as it is highly informative for various phenotypical characteristics. Here we aim to leverage metabolomics to impute missing data in clinical variables routinely assessed in large epidemiological and clinical studies.MethodsTo this end, we have employed ∼26,000 1H-NMR metabolomics samples from 28 Dutch cohorts collected within the BBMRI-NL consortium, to create 19 metabolomics-based predictors for clinical variables, including diabetes status (AUC5-Fold CV = 0·94) and lipid medication usage (AUC5-Fold CV = 0·90).FindingsSubsequent application in independent cohorts confirmed that our metabolomics-based predictors can indeed be used to impute a wide array of missing clinical variables from a single metabolomics data resource. In addition, application highlighted the potential use of our predictors to explore the effects of totally unobserved confounders in omics association studies. Finally, we show that our predictors can be used to explore risk factor profiles contributing to mortality in older participants.InterpretationTo conclude, we provide 1H-NMR metabolomics-based models to impute clinical variables routinely assessed in epidemiological studies and illustrate their merit in scenarios when phenotypic variables are partially incomplete or totally unobserved.FundingBBMRI-NL, X-omics, VOILA, Medical Delta and the Dutch Research Council (NWO-VENI).

Novel electrochemiluminescent assay for the aptamer-based detection of testosterone

This work presents a proof-of-concept assay for the detection and quantification of small molecules based on aptamer recognition and electrochemiluminescence (ECL) readout. The testosterone-binding (TESS.1) aptamer was used to demonstrate the novel methodology. Upon binding of the target, the TESS.1 aptamer is released from its complementary capture probe – previously immobilized at the surface of the electrode – producing a decrease in the ECL signal after a washing step removing the released (labeled) TESS.1 aptamer. The analytical capability of the ECL assay towards testosterone detection was investigated displaying a linear range from 0.39 to 1.56 μM with a limit of detection of 0.29 μM. The selectivity of the proposed assay was assessed by performing two different negative control experiments; i) detection of testosterone with a randomized ssDNA sequence and ii) detection of two other steroids, i.e. deoxycholic acid and hydrocortisone with the TESS.1 aptamer. In parallel, complementary analytical techniques were employed to confirm the suggested mechanism: i) native nano-electrospray ionization mass spectrometry (native nESI-MS) was used to determine the stoichiometry of the binding, and to characterize aptamer-target interactions; and, ii) isothermal titration calorimetry (ITC) was carried out to elucidate the dissociation constant (Kd) of the complex of testosterone and the TESS.1 aptamer. The combination of these techniques provided a complete understanding of the aptamer performance, the binding mechanism, affinity and selectivity. Furthermore, this important characterization carried out in parallel validates the real functionality of the aptamer (TESS.1) ensuring its use towards selective testosterone binding in further biosensors. This research will pave the way for the development of new aptamer-based assays coupled with ECL sensing for the detection of relevant small molecules.

Metabolomics datasets in the Born in Bradford cohort

Metabolomics is the quantification of small molecules, commonly known as metabolites. Collectively, these metabolites and their interactions within a biological system are known as the metabolome. The metabolome is a unique area of study, capturing influences from both genotype and environment. The availability of high-throughput technologies for quantifying large numbers of metabolites, as well as lipids and lipoprotein particles, has enabled detailed investigation of human metabolism in large-scale epidemiological studies. The Born in Bradford (BiB) cohort includes 12,453 women who experienced 13,776 pregnancies recruited between 2007-2011, their partners and their offspring. In this data note, we describe the metabolomic data available in BiB, profiled during pregnancy, in cord blood and during early life in the offspring. These include two platforms of metabolomic profiling: nuclear magnetic resonance and mass spectrometry. The maternal measures, taken at 26-28 weeks’ gestation, can provide insight into the metabolome during pregnancy and how it relates to maternal and offspring health. The offspring cord blood measurements provide information on the fetal metabolome. These measures, alongside maternal pregnancy measures, can be used to explore how they may influence outcomes. The infant measures (taken around ages 12 and 24 months) provide a snapshot of the early life metabolome during a key phase of nutrition, environmental exposures, growth, and development. These metabolomic data can be examined alongside the BiB cohorts’ extensive phenotype data from questionnaires, medical, educational and social record linkage, and other ‘omics data.

ImmunoAnalysis: A New Journal to Publish Peer-Reviewed Manuscripts in the Fields of Pharmaceutical Analysis and Immunology

The immunoassay area in clinical chemistry began with the development of the first immunoassay for insulin by Solomon Berson and Rosalyn S. Yalow in 1959. Their work resulted in their receipt of the Nobel Prize in Physiology or Medicine in 1977. The radioimmunoassay for insulin has paved the way for the development of immunoassays for thousands of other analytes over the ensuing half a century. A decision by Drs. Berson and Yalow to not patent this technology accelerated the progress of immunoassay development. Immunoassay technology continues to evolve with new applications and improved analytical platforms. The future appears to be headed in two directions: continued improvement in immunodetection methods for very high-sensitivity applications, and multiplex analysis. During 1995–2021, a wide range of immunoassays has been developed to provide the quantitative, semi-quantitative, or qualitative detection of analytes in various bioanalytical settings, such as clinical diagnostics, biopharmaceutical analysis, environmental monitoring, security, and food testing. However, there is only one specialized journal in this field namely "Journal of Immunoassay and Immunochemistry". ImmunoAnalysis as the second journal in this field will provide a platform for the publication of studies on all scientific aspects of immune analysis, including developing bioanalytical methods such as quantification of small molecules, peptides, proteins, antibodies, biomarkers, and immunoassay methods, and studies in immunology field such as innate immunity and inflammation; immune receptors, cellular and systemic immunity; vaccines; immune tolerance; autoimmunity, tumor immunology, and microbial immunopathology. It is a peer-reviewed, platinum open access journal (no processing or publication fees) intended to maintain the highest possible global scientific standards. We acknowledge the support from TUOMS press in making use of their facilities and for hosting the journal. The members of the editorial board by using their wide range of perspectives and expertise make a journal that will be an indispensable resource and a sum of parts offering something new to forthcoming studies. We have been buoyed and empowered by their supports. Crucially, of course, those reviewers who will spend their time and energy to assess submissions are a fundamental part of this endeavor. Finally, starting this journal is not meant to provide just another "outlet" for papers, but stems from the ambition to continue, develop and start important discussions in the field of immune analysis. We warmly invite you to join us as reviewers and authors to speed up the growth of this journal. The issues ImmunoAnalysis considers are yours.

Metabolomics in chronic pain research.

Metabolomics deals with the identification and quantification of small molecules (metabolites) in biological samples. As metabolite levels can reflect normal or altered metabolic pathways, their measurement provides information to improve the understanding, diagnosis and management of diseases. Despite its immense potential, metabolomics applications to pain research have been sparse. This paper describes current metabolomics techniques, reviews published human metabolomics pain research and compares successful metabolomics research in other areas of medicine with the goal of highlighting opportunities offered by metabolomics to advance pain medicine. Non-systematic review. Our search identified 19 studies that adopted a metabolomics approach in: fibromyalgia (7), chronic widespread pain (4), other musculoskeletal pain conditions (5), neuropathic pain (1), complex regional pain syndrome (1) and pelvic pain (1). The studies used either mass spectrometry or nuclear magnetic resonance. Most are characterized by small sample sizes. Some consistency has been found for alterations in glutamate and testosterone metabolism, and metabolic imbalances caused by the gut microbiome. Metabolomics research in chronic pain is in its infancy. Most studies are at the pilot stage. Metabolomics research has been successful in other areas of medicine. These achievements should motivate investigators to expand metabolomics research to improve the understanding of the basic mechanisms of human pain, as well as provide tools to diagnose, predict and monitor chronic pain conditions. Metabolomics research can lead to the identification of biomarkers to support the development and testing of treatments, thereby facilitating personalized pain medicine.

Metabolomics datasets in the Born in Bradford cohort.

Metabolomics is the quantification of small molecules, commonly known as metabolites. Collectively, these metabolites and their interactions within a biological system are known as the metabolome. The metabolome is a unique area of study, capturing influences from both genotype and environment. The availability of high-throughput technologies for quantifying large numbers of metabolites, as well as lipids and lipoprotein particles, has enabled detailed investigation of human metabolism in large-scale epidemiological studies. The Born in Bradford (BiB) cohort includes 12,453 women who experienced 13,776 pregnancies recruited between 2007-2011, their partners and their offspring. In this data note, we describe the metabolomic data available in BiB, profiled during pregnancy, in cord blood and during early life in the offspring. These include two platforms of metabolomic profiling: nuclear magnetic resonance and mass spectrometry. The maternal measures, taken at 26-28 weeks' gestation, can provide insight into the metabolome during pregnancy and how it relates to maternal and offspring health. The offspring cord blood measurements provide information on the fetal metabolome. These measures, alongside maternal pregnancy measures, can be used to explore how they may influence outcomes. The infant measures (taken around ages 12 and 24 months) provide a snapshot of the early life metabolome during a key phase of nutrition, environmental exposures, growth, and development. These metabolomic data can be examined alongside the BiB cohorts' extensive phenotype data from questionnaires, medical, educational and social record linkage, and other 'omics data.

SMfinder: Small Molecules Finder for Metabolomics and Lipidomics Analysis.

Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user-friendly open-source software, named SMfinder, for the robust identification and quantification of small molecules. The software introduces an MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and targeted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted, and flux analysis.

A novel fluorescent aptasensor for aflatoxin M1 detection using rolling circle amplification and g-C3N4 as fluorescence quencher

Aflatoxin M1 is a prevailing natural carcinogenic mycotoxin, found in milk. Herein, we reported a sensitive fluorescent aptasensor for AFM1, based on rolling circle amplification (RCA) technology to improve the sensitivity of the assay. The detection of AFM1 was achieved using KYF4: Eu3+ time-resolved fluorescent nanoparticles (KYF4: Eu3+ TRFNPs) as signal probe and graphitic carbon nitride (g-C3N4) nanosheet as a quencher. The AFM1 aptamer act as a recognition element and as a primer for DNA synthesis. TRFNPs attached to complementary DNA (TRFNPs-cDNA) acts as a signal probe. In the absence of target, a rolling circle template (RCT) ligated to aptamer and RCA reaction initiated. The TRFNPs-cDNA was strengthened to amplified RCA product (RCAP) to make RCAP/TRFNPs-cDNA duplex. This duplex cannot be adsorbed onto g-C3N4, and ultimately no fluorescence quenched. In the presence of AFM1, aptamer recognized the target and aptamer-target complex was formed. Hence, no aptamer hybridized with RCT, therefore, no RCA and duplex formation. Consequently, free TRFNPs-cDNA adsorbed onto g-C3N4 and resulted in quenched fluorescence. The assay provides a lower detection limit (0.0194 pg/mL) than the previously reported assays and has good recoveries between 92–99.8 %. Hence, the present method would be a suitable method for the quantification of small molecules.

Graphene/aptamer probes for small molecule detection: from in vitro test to in situ imaging.

Small molecules are key targets in molecular biology, environmental issues, medicine and food industry. However, small molecules are challenging to be detected due to the difficulty of their recognition, especially in complex samples, such as in situ in cells or animals. The emergence of graphene/aptamer probes offers an excellent opportunity for small molecule quantification owing to their appealing attributes such as high selectivity, sensitivity, and low cost, as well as the potential for probing small molecules in living cells or animals. This paper (with 130 refs.) will review the application of graphene/aptamer probes for small molecule detection. We present the recent progress in the design and development of graphene/aptamer probes enabling highly specific, sensitive and rapid detection of small molecules. Emphasis isplaced on the success in their development andapplication for monitoring small molecules in living cells and in vivo systems. By discussing the key advances in this field, we wish to inspire more research work of the development of graphene/aptamer probes for both on-site or in situ detection of small molecules and its applications for investigating the functions of small molecules in cells in a dynamic way. Graphical abstract Graphene/aptamer probes can be used to construct different platforms for detecting small molecules with high specificity and sensitivity, both in vitro and in situ in living cells and animals.

Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics.

Vendor-independent software tools for quantification of small molecules and metabolites are lacking, especially for targeted analysis workflows. Skyline is a freely available, open-source software tool for targeted quantitative mass spectrometry method development and data processing with a 10 year history supporting six major instrument vendors. Designed initially for proteomics analysis, we describe the expansion of Skyline to data for small molecule analysis, including selected reaction monitoring, high-resolution mass spectrometry, and calibrated quantification. This fundamental expansion of Skyline from a peptide-sequence-centric tool to a molecule-centric tool makes it agnostic to the source of the molecule while retaining Skyline features critical for workflows in both peptide and more general biomolecular research. The data visualization and interrogation features already available in Skyline, such as peak picking, chromatographic alignment, and transition selection, have been adapted to support small molecule data, including metabolomics. Herein, we explain the conceptual workflow for small molecule analysis using Skyline, demonstrate Skyline performance benchmarked against a comparable instrument vendor software tool, and present additional real-world applications. Further, we include step-by-step instructions on using Skyline for small molecule quantitative method development and data analysis on data acquired with a variety of mass spectrometers from multiple instrument vendors.

Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?

While mass spectrometry (MS)-based quantification of small molecules has been successfully used for decades, targeted MS has only recently been used by the proteomics community to investigate clinical questions such as biomarker verification and validation. Targeted MS holds the promise of a paradigm shift in the quantitative determination of proteins. Nevertheless, targeted quantitative proteomics requires improvisation in making sample processing, instruments, and data analysis more accessible. In the backdrop of the genomic era reaching its zenith, certain questions arise: is the proteomic era about to come? If we are at the beginning of a new future for protein quantification, are we prepared to incorporate targeted proteomics at the benchside for basic research and at the bedside for the good of patients? Here, an overview of the knowledge required to perform targeted proteomics as well as its applications is provided. A special emphasis is placed on upcoming areas such as peptidomics, proteoform research, and mass spectrometry imaging, where the utilization of targeted proteomics is expected to bring forth new avenues. The limitations associated with the acceptance of this technique for mainstream usage are also highlighted. Also see the video abstract here https://youtu.be/mieB47B8gZw.

Metabolomic Applications in Radiation Biodosimetry.

Metabolomics, the collective assessment and quantification of small molecules in a given biofluid or tissue sample, has provided new ways in evaluating an individual's exposure level to ionizing radiation or other genotoxic stressors. Protocols that are routinely utilized for the preparation of samples from rodents to patients are presented here in order to be analyzed by high-throughput liquid chromatography-mass spectrometry techniques. These protocols are based on established methods in our laboratory that have been used extensively in radiation biodosimetry through metabolomics. These protocols are focused on general profiling of samples and therefore do not concentrate on extraction of specific classes on metabolites (e.g., eicosanoids).