Mass Spectrometry Techniques Research Articles

Quantification of N-acetyl-l-aspartate in dried blood spots: A simple and fast LC-MS/MS neonatal screening method for the diagnosis of Canavan disease

BackgroundCanavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. MethodsWe therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. ResultsMeasuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6–13.6 μmol/L [n = 45] vs 0.44; 0.24–0.99 μmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1–9.9 μmol/L [n = 8]) and neonatal controls (0.93; 0.4–1.8 μmol/L [n = 784]) (p < 0.05). ConclusionsWe have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.

Chemical profile, anticholinesterase and molecular docking studies of Boesenbergia albosanguinea (Ridl.) Loes. essential oil

Boesenbergia albosanguinea is a member of the genus Boesenbergia, which traditionally serves a range of purposes, including food, medicinal applications, ornamental use, and ritualistic practices. It is found growing in wet, shady forest areas on sandstone or quartz-derived soils. This study aims to characterize the essential oils from the rhizomes of B. albosanguinea, assessing their activity against acetylcholinesterase using in vitro methods, along with molecular docking analysis. The essential oil was obtained through hydrodistillation and the volatile components were analysed using gas chromatography (GC-FID) and gas chromatography mass spectrometry (GC-MS) techniques. A total of 34 chemical components, which constitute 96.7% of the oil content, were successfully identified. The most abundant components were elemicin (44.0%), α-gurjunene (9.3%), β-caryophyllene (4.5%), and safrole (4.1%). Anticholinesterase activity was assessed using Ellman method. A moderate inhibitory effect was observed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes with IC50 values of 90.5 ± 0.2 and 110.8 ± 0.2 μg/mL, respectively. The molecular docking studies of elemicin against the cholinesterase target revealed a robust docking score (-7.0 kcal/mol), but it also showed important interactions with some key amino acid residues, providing crucial insights into its potential as a cholinesterase inhibitor.

Surface assisted laser desorption/ionization mass spectrometry of lithium cationized lignans – A tool for rapid screening of plant extracts

Lignans are one of the most widespread classes of plant secondary metabolites possessing unique biological activities. The search for new sources of such compounds, their screening and identification in plant biomass is an urgent task, requiring the use of advanced analytical methods. In the present study, matrix-free surface assisted laser desorption/ionization (SALDI) mass spectrometry was proposed for its solution. The combination of carbon nanocoating sputtered on a target plate with lithium cationization allowed obtaining high-quality mass spectra of eight coniferous wood lignans with different bonds between aromatic units (secoisolariciresinol, matairesinol, 7-hydroxymatairesinol, nortrachelogenin, pinoresinol, guaiacylglycerol-β-guaiacyl ether, dehydrodihydrodiisoeugenol, and divanillin). Three types of fragmentation methods (post-source decay, high- and low-energy collision induced dissociation) for the first time were implemented on two types of MALDI instruments. Regardless of tandem mass spectrometry technique, lignans demonstrated the similar fragmentation patterns including the cleavage of bonds between aromatic units with the formation of mostly lithiated product ions. The low-energy collision induced dissociation in quadrupole ion trap provided the most informative tandem mass spectra and multi-stage fragmentation capabilities in structural studies. On this basis, an approach to rapid, high-throughput, and low-cost screening and tentative identification of lignans including isomeric compounds in plant biomass was developed and successfully tested on real samples of pine, fir, spruce, and larch knotwood extracts.

Mass Spectrometry-Based Analyses of Carbon Nanodots: Structural Elucidation.

Carbon nanodots (CNDs) are nanomaterials with ubiquitous applications in health for diagnosis and treatments. The key to enhancing the applications of carbon nanodots in various fields lies on how deep its structure is understood. Here, we review the mass spectroscopy (MS) techniques employed for carbon nanodot analysis. We aimed to revive the use of MS to support the structural elucidation of carbon nanodots. General techniques used in nanomaterials characterization include laser desorption/ionization (LDI), matrix-assisted LDI (MALDI), inductively coupled plasma (ICP), and electrospray ionization (ESI) MS. For CNDs characterization, LDI-MS, MALDI-MS, and ESI-MS were employed. The techniques required further instrumentations of time-of-flight (TOF), for MALDI, and TOF, quadrupole (Q), and tandem (MS/MS) for ESI. LDI-MS could be applied to prove the surface and core structural composition of carbon nanodots. Meanwhile, MALDI-MS was used to elucidate the surface structures of CNDs. Finally, ESI-MS could provide significant insight into the carbon nanodots' structural composition and bonding patterns. In summary, MS could be combined with other techniques to unambiguously elucidate the structure of carbon nanodots.

Elucidating hormone transduction and the protein response of soybean roots to drought stress based on ultra performance liquid chromatography–tandem mass spectrometry and four-dimensional data-independent acquisition

Drought stress can affect the growth and development of soybean, an important cultivated crop. As the main water-absorbing organ, it is particularly important to study the drought response mechanism of roots. Plant hormones are associated with almost all basic biological processes and are used as candidate targets for improving plant stress resistance by regulating cell signal transduction related to stress resistance. In this study, ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) and four-dimensional data-independent (4D-DIA) techniques were used to study the root hormone changes (5 %, 10 %, 15 % and 20 % drought stress level) and proteomic responses (15 % drought stress level) of different drought-tolerant varieties (drought-resistant HN44 and drought-sensitive HN65) under drought stress. The root activity, CAT activity and soluble sugar content of HN44 were basically higher than those of HN65 under different drought stress levels and days. In the two varieties, some plant hormones, such as abscisic acid (ABA) and salicylic acid (SA), accumulated with an increase in drought degree, and the accumulation of these hormones can give plants a certain degree of drought resistance. Most of the plant hormones peaked at 5–15 % drought stress level, and the overall content of jasmonic acid (JA) and cytokinin (CKs) decreased. Differences in the expression of key enzymes involved in hormone signal transduction, such as GH3, TCH4, and PR1, affect a variety of processes and lead to differences in drought resistance between the two varieties. In addition, HN44 had more upregulated proteins in important antioxidant pathways such as glutathione metabolism, phenylpropanoid biosynthesis, and isoflavone biosynthesis. Important drought-responsive proteins, such as PAL, 4CL, and VR, have also been characterized in HN44. In general, the drought-resistant variety was substantially better than the drought-sensitive variety in terms of hormone signaling and antioxidant capacity. These candidate proteins can be used as potential targets for improving drought resistance.

Exploring free amino acid profiles in CCHF patients: Implications for disease progression.

This study investigated the intricate interplay between Crimean-Congo hemorrhagic fever virus (CCHFV) infection and alterations in amino acid metabolism. Our primary aim is to elucidate the impact of Crimean-Congo hemorrhagic fever (CCHF) on specific amino acid concentrations and identify potential metabolic markers associated with viral infection. One hundred ninety individuals participated in this study, comprising 115 CCHF patients, 30 CCHF negative patients, and 45 healthy controls. Liquid chromatography-tandem mass spectrometry techniques were employed to quantify amino acid concentrations. The amino acid metabolic profiles in CCHF patients exhibit substantial distinctions from those in the control group. Patients highlight distinct metabolic reprogramming, notably characterized by arginine, histidine, taurine, glutamic acid, and glutamine metabolism shifts. These changes have been associated with the underlying molecular mechanisms of the disease. Exploring novel therapeutic and diagnostic strategies addressing specific amino acids may offer potential means to mitigate the severity of the disease.

Fungal-bacteria interactions provide shelter for bacteria in Caesarean section scar diverticulum.

Caesarean section scar diverticulum (CSD) is a significant cause of infertility among women who have previously had a Caesarean section, primarily due to persistent inflammatory exudation associated with this condition. Even though abnormal bacterial composition is identified as a critical factor leading to this chronic inflammation, clinical data suggest that a long-term cure is often unattainable with antibiotic treatment alone. In our study, we employed metagenomic analysis and mass spectrometry techniques to investigate the fungal composition in CSD and its interaction with bacteria. We discovered that local fungal abnormalities in CSD can disrupt the stability of the bacterial population and the entire microbial community by altering bacterial abundance via specific metabolites. For instance, Lachnellula suecica reduces the abundance of several Lactobacillus spp., such as Lactobacillus jensenii, by diminishing the production of metabolites like Goyaglycoside A and Janthitrem E. Concurrently, Clavispora lusitaniae and Ophiocordyceps australis can synergistically impact the abundance of Lactobacillus spp. by modulating metabolite abundance. Our findings underscore that abnormal fungal composition and activity are key drivers of local bacterial dysbiosis in CSD.

Fungal–bacteria interactions provide shelter for bacteria in Caesarean section scar diverticulum

Caesarean section scar diverticulum (CSD) is a significant cause of infertility among women who have previously had a Caesarean section, primarily due to persistent inflammatory exudation associated with this condition. Even though abnormal bacterial composition is identified as a critical factor leading to this chronic inflammation, clinical data suggest that a long-term cure is often unattainable with antibiotic treatment alone. In our study, we employed metagenomic analysis and mass spectrometry techniques to investigate the fungal composition in CSD and its interaction with bacteria. We discovered that local fungal abnormalities in CSD can disrupt the stability of the bacterial population and the entire microbial community by altering bacterial abundance via specific metabolites. For instance, Lachnellula suecica reduces the abundance of several Lactobacillus spp., such as Lactobacillus jensenii, by diminishing the production of metabolites like Goyaglycoside A and Janthitrem E. Concurrently, Clavispora lusitaniae and Ophiocordyceps australis can synergistically impact the abundance of Lactobacillus spp. by modulating metabolite abundance. Our findings underscore that abnormal fungal composition and activity are key drivers of local bacterial dysbiosis in CSD.

Exploring free amino acid profiles in Crimean-Congo hemorrhagic fever patients: Implications for disease progression.

This study investigated the intricate interplay between Crimean-Congo hemorrhagic fever virus infection and alterations in amino acid metabolism. The primary aim is to elucidate the impact of Crimean-Congo hemorrhagic fever (CCHF) on specific amino acid concentrations and identify potential metabolic markers associated with viral infection. One hundred ninety individuals participated in this study, comprising 115 CCHF patients, 30 CCHF negative patients, and 45 healthy controls. Liquid chromatography-tandem mass spectrometry techniques were employed to quantify amino acid concentrations. The amino acid metabolic profiles in CCHF patients exhibit substantial distinctions from those in the control group. Patients highlight distinct metabolic reprogramming, notably characterized by arginine, histidine, taurine, glutamic acid, and glutamine metabolism shifts. These changes have been associated with the underlying molecular mechanisms of the disease. Exploring novel therapeutic and diagnostic strategies addressing specific amino acids may offer potential means to mitigate the severity of the disease.

Quinoxaline clubbed thiazole: Molecular docking, synthesis and antimicrobial evaluation

Novel hybrid molecules of thiazole with quinoxaline have been designed, synthesized and characterised by FT-IR, Mass, 1H and 13C NMR spectroscopic techniques. The pharmacological potential of synthesized compounds was checked for bacterial and fungal strains by the Broth microdilution method. All molecules exhibited significant antibacterial and antifungal activities. The compound (E)-4-(4-chlorophenyl)-2-(2-(1-(3-methylquinoxalin-2-yl)ethylidene)hydrazinyl)thiazole (3b) displayed (MIC = 12.5 μg/mL) excellent biological potential against Staphylococcus aureus [MTCC96] gram-positive bacterial strain. The molecular docking of thiazole derivatives was performed with S. aureus DNA gyrase (2XCT). The docked molecules displayed significant interactions with the active cavity of S. aureus DNA gyrase. The (E)-4-(4-chlorophenyl)-2-(2-(1-(3-methylquinoxalin-2-yl)ethylidene)hydrazinyl)thiazole (3b) molecule displayed the most stable conformation at the highest binding affinity (−10.9 kcal/mol) among the docked molecules with protein.

Genesis of Cu-Sn Mineralization in the Shuangjianzishan Super-Large Silver Deposit, Inner Mongolia: Trace Element Constraints from Chalcopyrite and Cassiterite

The Shuangjianzishan silver polymetallic deposit is located in the copper–tin–lead–zinc–silver polymetallic metallogenic belt in the Southern Great Xing’an Range, with silver resources of more than 18,000 t, which is the largest silver polymetallic deposit in Asia. Early studies concluded that the Shuangjianzishan deposit is typically an epithermal Ag-Pb-Zn deposit that lacks a high-temperature mineralization stage. In recent years, with the deepening of research, a large amount of Cu-Sn mineralization has been found in the deep part of the Shuangjianzishan deposit, but it is less studied. The laser-ablation inductively coupled mass spectroscopy (LA-ICP-MS) technique is used to investigate the distribution and substitution of trace elements in chalcopyrite and cassiterite. In this paper, the trace element study of chalcopyrite and cassiterite from the Shuangjianzishan deposit reveals that Sn, In, As, Se, Sb, and Tl mainly exist in chalcopyrite in isomorphic form, while Pb, Bi, and Ni mainly exist in chalcopyrite in the form of mineral inclusions. The enrichment of the high-temperature elements Sn and Se in chalcopyrite, and the deficit of the middle- and low-temperature elements Ga, Sb, etc., reflect that the chalcopyrite in the Shuangjianzishan deposit was formed in a middle- and high-temperature environment, and it also indicates that the early ore-forming hydrothermal solution may be rich in Sn. Fe, In, Co, and Ni mainly exist in cassiterite in isomorphic form, and the content of W in cassiterite is high. There are two main forms, one is isomorphic and the other is wolframite inclusion. Cassiterite has Fe-rich and W-U-poor characteristics, indicating that cassiterite from the Shuangjianzishan deposit was formed under relatively oxidized conditions, and the relative enrichment of elements such as Fe, W, Zr, and Hf indicates that the temperature of cassiterite formation was high. The elemental content and inter-ionic coupling relationships suggest that the cassiterite from the Shuangjianzishan deposit may have an elemental replacement mechanism of W6+ + Fe2+ ↔ 2Sn4+ or Fe3+ + OH− ↔ Sn4+ + O2−. The trace elements in cassiterite of the Shuangjianzishan deposit are rich in Fe and Mn and depleted in Nb and Ta, according to the Fe-W diagram, and the tin mineralization of the Shuangjianzishan deposit belongs to cassiterite–sulfide-type tin mineralization. Chalcopyrite Co/Ni ratios &gt;1 are consistent with the characteristics of chalcopyrite genesis in hydrothermal deposits.

An innovative “Up-and-Down” adsorption processes for pyrene removal from acid wastewater as new approach in water remediation

Polycyclic Aromatic Hydrocarbons (PAH) pose a risk to human health because they are highly toxic, carcinogenic, and mutagenic. In the context of water remediation, hybrid biopolymers are green materials that can reduce the pollutants in environmental matrices. This study aims to propose a new “up-and-down” wastewater treatment system based on the adsorption of contaminants from the bulk and their transport to the surface to promote future remediation processes. Pyrene was selected as a model pollutant for PAH, and hybrid alginate beads, such as thermally modified hematite montmorillonite alginate calcium carbonate beads (@H@MACaBs), were prepared to investigate their use in water remediation. The synthesized beads were characterized using FT-IR SEM and ion-TOF mass spectrometry techniques, and adsorption studies were performed to quantify the remediation process efficiency and investigate the adsorption processes. Approximately 50 % of pyrene was removed from the aqueous solution without stirring after 90 min of contact. A double increase in pyrene removal was observed when the classical hybrid alginate beads were compared with the new modified hybrid alginate beads. This study contributes to efforts to deploy innovative remediation strategies to reduce the environmental impact of the textile industry, which is a significant contributor to global manufacturing and pollution.

Modern Electrospray Ionization Mass Spectrometry Techniques for the Characterization of Supramolecules and Coordination Compounds.

Mass spectrometry is routinely used for myriad applications in clinical, industrial, and research laboratories worldwide. Developments in the areas of ionization sources, high-resolution mass analyzers, tandem mass spectrometry, and ion mobility have significantly extended the repertoire of mass spectrometrists; however, for coordination compounds and supramolecules, mass spectrometry remains underexplored and arguably underappreciated. Here, the reader is guided through different tools of modern electrospray ionization mass spectrometry that are suitable for larger inorganic complexes. All steps, from sample preparation and technical details to data analysis and interpretation are discussed. The main target audience of this tutorial is synthetic chemists as well as technicians/mass spectrometrists with little experience in characterizing labile inorganic compounds.

Rapid analysis of the chemical constituents of traditional Tibetan medicine Sbyor-bzo-ghi-wang using ballpoint electrospray ionization technique.

The chemical constituents of traditional Tibetan medicines (TTM) can be identified using high-performance liquid chromatography and high-resolution mass spectrometry (HPLC-MS/MS) technique. However, the HPLC-MS/MS technique requires the sample to be pretreated and then separated using the specific liquid chromatography method, which is time consuming. This study developed a ballpoint electrospray ionization (BPESI) technique for analyzing the chemical constituents of Sbyor-bzo-ghi-wang. This technique is a simple and inexpensive method for the rapid identification of the chemical constituents of TTMs. After the important parameters of the homemade BPESI device were optimized, the chemical constituents of Sbyor-bzo-ghi-wang were quickly identified without sample pretreatment. The raw data were converted to mzML file using MSConvert and then identified using SIRIUS 5 software. The results showed that 30 compounds were identified from Sbyor-bzo-ghi-wang, namely eight bile acids, six flavonoids, four alkaloids, three amino acids, and nine others. Compared to the ultra-high-performance liquid chromatography-Q/Orbitrap and high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS) technique, the BPESI technique identified almost similar types of compounds and also a comparable number of compounds. Compared with the traditional HPLC-MS/MS methods, the BPESI technique does not require complex sample pretreatment and subsequent chromatographic separation steps; also it consumes a small quantity of samples. Therefore, BPESI can be used for the qualitative analysis of the chemical constituents of Sbyor-bzo-ghi-wang.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Highly stable Ag-doped Cu2O immobilized cellulose-derived carbon beads with enhanced visible-light photocatalytic degradation of levofloxacin

Ag-doped Cu2O immobilized carbon beads (Ag/Cu2O@CB) based composite photocatalysts have been prepared for the removal of levofloxacin, an antibiotic, from water. The photocatalysts were prepared by the processes of chemical reduction and in-situ solid-phase precipitation. The composite photocatalyst was characterized by a porous and interconnected network structure. Ag nanoparticles were deposited on Cu2O particles to develop a metal-based semiconductor to increase the catalytic efficiency of the system and the separation efficiency of the photogenerated carriers. Cellulose-derived carbon beads (CBs) can also be used as electron storage libraries which can capture electrons released from the conduction band of Cu2O. The results revealed that the maximum catalytic degradation efficiency of the composite photocatalyst for the antibiotic levofloxacin was 99.02 %. The Langmuir–Hinshelwood model was used to study the reaction kinetics, and the process of photodegradation followed first-order kinetics. The maximum apparent rate was recorded to be 0.0906 min-1. The mass spectrometry technique showed that levofloxacin degraded into carbon dioxide and water in the presence of the photocatalyst. The results revealed that the easy-to-produce photocatalyst was stable and efficient in levofloxacin removing.

New silver, rhodium and iridium complexes with anthracene-functionalized N-heterocyclic carbene ligands: Crystal structures, cytotoxicity and fluorescence studies

In the present study, we have synthesized anthracene-functionalized metal complexes to investigate their cytotoxicity and photophysical properties. In this context, firstly, an Ag–NHC (NHC = N-heterocyclic carbene) complex (2a) has been synthesized by the interaction of Ag2O and N-(2-methylbenzyl)-N-((anthracen-9-yl)methyl)benzimidazolium chloride (1a). Corresponding Rh– (3a) and Ir–NHC (4a) complexes have also been synthesized by the transmetalation reaction between 2a and corresponding metal compounds. Additionally, for comparison purposes, an ether-functionalized Ag–NHC complex (2b) with N-(methoxyethyl)-N-((anthracene-9-yl)methyl)benzimidazole-2-ylidene ligand, and [AgL2]NO3 type complex (2c) with a N-coordinated N-((anthracen-9-yl)methyl)benzimidazole ligand have been synthesized. All complexes have been characterized by the combination of NMR and mass spectroscopic techniques, and elemental analyses. Moreover, solid state structures of 2a, 2b and 4a have been determined by the single crystal X-ray diffraction analysis. Cytotoxic potentials of all compounds have been tested against human lung adenocarcinoma alveolar basal epithelial cell line (A549) using the MTT assay, and all complexes performed strong anti-proliferative activity. Stability studies have disclosed that only 1a and 2c are able to retain their structure in the culture medium (DMEM) that biological assays were carried out. Photophysical measurements revealed that all compounds have similar emission bands because of the conjugation of anthracene group. Finally, 1a and 2c have been visualized by fluorescence confocal microscopy to investigate cellular uptake and subcellular distribution of the complexes, and the findings indicated that both compounds dispersed along the cell membranes nuclear regions.

Distyryl Carboxylic Acid Substituted BODIPY and BODIPY‐C60 Systems for Generation of Singlet Oxygen

AbstractBODIPY based molecules functionalized with water soluble moieties have been developed and recognized as liable triplet photosensitizers for phototherapeutic applications. In this work, we have prepared BODIPY analogues with dicarboxylic acid groups and bearing iodine or fullerene moieties for efficient intersystem crossing which display far‐red absorption band and high singlet oxygen production both in organic and aqueous media. In this perspective by using similar design the effect of the halogenation of the dyes versus spin converter on the skeleton were compared. The structures of the synthesized BODIPYs and BODIPY‐C60 (6–10) were elucidated by mass, 1H and 13C NMR spectroscopic techniques. Photophysical and photochemical properties of the compounds were examined using UV‐Vis. absorption and fluorescence emission spectrophotometers. The absorption intensities of the trap molecules (DPBF and ADBA) reduced because of singlet oxygen generation in the media. This study may provide useful infrastructure to elucidate the relationship between the use of fullerene (9) or iodine (10) where singlet oxygen quantum yield of 9 and 10 are determined as 0.87 and 0.97 respectively.

A guide to precise measurements of isotope abundance by ESI-Orbitrap MS.

Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization-Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules ('isotopocules'). This protocol describes two procedures. In the first one, we provide a step-by-step beginner's guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2-3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology.

Association between visceral adipose tissue and total testosterone among the United States male adults: a cross-sectional study.

Visceral adipose tissue (VAT) is regarded as an important risk factor for obesity-related diseases. The results of the association between VAT and total testosterone (TT) are controversial and whether this association is nonlinear is still unknown. 3971 male participants who were aged 20-59 years from the National Health and Nutrition Examination Surveys 2011-2016 were included. VAT area was measured by dual-energy x-ray absorptiometry. TT in serum was assessed utilizing the isotope dilution liquid chromatography-tandem mass spectrometry technique. Linear regression models assessed the associations between VAT area and TT. A restricted cubic spline model was employed to investigate nonlinear relationships. A two-piecewise linear regression model was applied to determine the threshold effect. Subgroup analyses were conducted. The weighted methods were utilized in all analyses. VAT area was inversely associated with TT in the crude and adjusted models. In the fully adjusted model, VAT area was associated with TT (β = -0.59, 95% confidence interval [CI] = -0.74, -0.43) and compared to the first tertile of VAT area, the second and the third tertile had a lower TT level, the β and 95% CI = -65.49 (-83.72, -47.25) and -97.57 (-121.86, -73.27) respectively. We found these inverse associations were nonlinear. The cutoff point of the VAT area was 126 cm2. When the VAT area was <126 cm2, VAT area was significantly associated with a lower TT level (β = -1.55, 95% CI = -1.93 to -1.17, p < 0.0001). However, when the VAT area was ≥126 cm2, this association was less apparent (β = -0.26, 95% CI = -0.52 to 0.01, p = 0.06). No significant interactions among different ages (<50 or ≥50 years), marital, and physical activity status were found. These findings underscore the potential for VAT area as a modifiable indicator for improving testosterone deficiency.