Liquid Chromatography Ion Mobility Mass Research Articles

Comprehensive Steroid Assay with Non-Targeted Analysis Using Liquid Chromatography Ion Mobility Mass Spectrometry.

Aldosterone-producing adenomas (APAs) have different steroid profiles in serum, depending on the causative genetic mutation. Ion mobility is a separation technique for gas-phase ions based on their m/z values, shapes, and sizes. Human serum (100 µL) was purified by liquid-liquid extraction using tert-butyl methyl ether/ethyl acetate at 1/1 (v/v) and mixed with deuterium-labeled steroids as the internal standard. The separated supernatant was dried, re-dissolved in water containing 20% methanol, and injected into a liquid chromatography-ion mobility-mass spectrometer (LC/IM/MS). We established a highly sensitive assay system by separating 20 steroids based on their retention time, m/z value, and drift time. Twenty steroids were measured in the serum of patients with primary aldosteronism, essential hypertension, and healthy subjects and were clearly classified using principal component analysis. This method was also able to detect phosphatidylcholine and phosphatidylethanolamine, which were not targeted. LC/IM/MS has a high selectivity for known compounds and has the potential to provide information on unknown compounds. This analytical method has the potential to elucidate the pathogenesis of APA and identify unknown steroids that could serve as biomarkers for APA with different genetic mutations.

A Novel Approach to Characterize the Lipidome of Marine Archaeon Nitrosopumilus maritimus by Ion Mobility Mass Spectrometry.

Archaea are differentiated from the other two domains of life by their biomolecular characteristics. One such characteristic is the unique structure and composition of their lipids. Characterization of the whole set of lipids in a biological system (the lipidome) remains technologically challenging. This is because the lipidome is innately complex, and not all lipid species are extractable, separable, or ionizable by a single analytical method. Furthermore, lipids are structurally and chemically diverse. Many lipids are isobaric or isomeric and often indistinguishable by the measurement of mass or even their fragmentation spectra. Here we developed a novel analytical protocol based on liquid chromatography ion mobility mass spectrometry to enhance the coverage of the lipidome and characterize the conformations of archaeal lipids by their collision cross-sections (CCSs). The measurements of ion mobility revealed the gas-phase ion chemistry of representative archaeal lipids and provided further insights into their attributions to the adaptability of archaea to environmental stresses. A comprehensive characterization of the lipidome of mesophilic marine thaumarchaeon, Nitrosopumilus maritimus (strain SCM1) revealed potentially an unreported phosphate- and sulfate-containing lipid candidate by negative ionization analysis. It was the first time that experimentally derived CCS values of archaeal lipids were reported. Discrimination of crenarchaeol and its proposed stereoisomer was, however, not achieved with the resolving power of the SYNAPT G2 ion mobility system, and a high-resolution ion mobility system may be required for future work. Structural and spectral libraries of archaeal lipids were constructed in non-vendor-specific formats and are being made available to the community to promote research of Archaea by lipidomics.

Characterization of the Exometabolome of Nitrosopumilus maritimus SCM1 by Liquid Chromatography-Ion Mobility Mass Spectrometry.

Marine Thaumarchaeota (formerly known as the marine group I archaea) have received much research interest in recent years since these chemolithoautotrophic organisms are abundant in the subsurface ocean and oxidize ammonium to nitrite, which makes them a major contributor to the marine carbon and nitrogen cycles. However, few studies have investigated the chemical composition of their exometabolome and their contributions to the pool of dissolved organic matter (DOM) in seawater. This study exploits the recent advances in ion mobility mass spectrometry (IM-MS) and integrates this instrumental capability with bioinformatics to reassess the exometabolome of a model ammonia-oxidizing archaeon, Nitrosopumilus maritimus strain SCM1. Our method has several advantages over the conventional approach using an Orbitrap or ion cyclotron resonance mass analyzer and allows assignments or annotations of spectral features to known metabolites confidently and indiscriminately, as well as distinction of biological molecules from background organics. Consistent with the results of a previous report, the SPE-extracted exometabolome of N. maritimus is dominated by biologically active nitrogen-containing metabolites, in addition to peptides secreted extracellularly. Cobalamin and associated intermediates, including α-ribazole and α-ribazole 5′-phosphate, are major components of the SPE-extracted exometabolome of N. maritimus. This supports the proposition that Thaumarchaeota have the capacity of de novo biosynthesizing cobalamin. Other biologically significant metabolites, such as agmatidine and medicagenate, predicted by genome screening are also detected, which indicates that Thaumarchaeota have remarkable metabolic potentials, underlining their importance in driving elemental cycles critical to biological processes in the ocean.

Clustering and Nonclustering Modifier Mixtures in Differential Mobility Spectrometry for Multidimensional Liquid Chromatography Ion Mobility-Mass Spectrometry Analysis.

Modifiers provide fast and reliable tuning of separation in differential mobility spectrometry (DMS). DMS selectivity for separating isomeric molecules depends on the clustering modifier concentration, which is typically 1.5-3 mol % ratio of isopropanol or ethanol in nitrogen. Low concentrations (0.1%) of isopropanol were found to improve resolution and sensitivity but at the cost of practicality and robustness. Replacing the single-channel DMS pump with a binary high-performance liquid chromatography (HPLC) pump enabled the generation of modifier mixtures at a constant flow rate using an isocratic or gradient mode, and the analytical benefits of the system were investigated considering cyclohexane, n-hexane, or n-octane as nonclustering modifiers and isopropanol or ethanol as clustering modifiers. It was found that clustering and nonclustering modifier mixtures enable optimization of selectivity, resolution, and sensitivity for different positional isomers and diastereoisomers. Data further suggested different ion separation mechanisms depending on the modifier ratios. For 85 analytes, the absolute difference in compensation voltages (CoVs) between pure nitrogen and cyclohexane at 1.5 mol % ratio was below 4 V, demonstrating its potential as a nonclustering modifier. Cyclohexane's nonclustering behavior was further supported by molecular modeling using density functional theory (DFT) and calculated cluster binding energies, showing positive ΔG values. The ability to control analyte CoVs by adjusting modifier concentrations in isocratic and gradient modes is beneficial for optimizing multidimensional LCxDMS-MS. It is fast and effective for manipulating the DMS scanning window size to realize shorter mass spectrometry (MS) acquisition cycle times while maintaining a sufficient number of CoV steps and without compromising DMS separation performance.

A Workflow for the Identification of Mycotoxin Metabolites Using Liquid Chromatography-Ion Mobility-Mass Spectrometry.

The structural identification of phase-I and phase-II metabolites of mycotoxins is a difficult task, mostly due to the lack of standards and because of the large number of isomeric forms. Here, we describe the use of ion mobility-mass spectrometry to analyze cereal extracts and how structural information on newly discovered mycotoxins metabolites could be obtained.

High-throughput serum proteomics for the identification of protein biomarkers of mortality in older men.

SummaryThe biological perturbations associated with incident mortality are not well elucidated, and there are limited biomarkers for the prediction of mortality. We used a novel high‐throughput proteomics approach to identify serum peptides and proteins associated with 5‐year mortality in community‐dwelling men age ≥65 years who participated in a longitudinal observational study of musculoskeletal aging (Osteoporotic Fractures in Men: MrOS). In a discovery phase, serum specimens collected at baseline in 2473 men were analyzed using liquid chromatography–ion mobility–mass spectrometry, and incident mortality in the subsequent 5 years was ascertained by tri‐annual questionnaire. Rigorous statistical methods were utilized to identify 56 peptides (31 proteins) that were associated with 5‐year mortality. In an independent replication phase, selected reaction monitoring was used to examine 21 of those peptides in baseline serum from 750 additional men; 81% of those peptides remained significantly associated with mortality. Mortality‐associated proteins included a variety involved in inflammation or complement activation; several have been previously linked to mortality (e.g., C‐reactive protein, alpha 1‐antichymotrypsin) and others are not previously known to be associated with mortality. Other novel proteins of interest included pregnancy‐associated plasma protein, VE‐cadherin, leucine‐rich α‐2 glycoprotein 1, vinculin, vitronectin, mast/stem cell growth factor receptor, and Saa4. A panel of peptides improved the predictive value of a commonly used clinical predictor of mortality. Overall, these results suggest that complex inflammatory pathways, and proteins in other pathways, are linked to 5‐year mortality risk. This work may serve to identify novel biomarkers for near‐term mortality.

In Utero Exposure to Histological Chorioamnionitis Primes the Exometabolomic Profiles of Preterm CD4+ T Lymphocytes.

Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the risk for preterm birth, death, and disability because of persistent systemic and localized inflammation. The immunological mechanisms sustaining this response in the preterm newborn remain unclear. We sought to determine the consequences of HCA exposure on the fetal CD4+ T lymphocyte exometabolome. We cultured naive CD4+ T lymphocytes from HCA-positive and -negative preterm infants matched for gestational age, sex, race, prenatal steroid exposure, and delivery mode. We collected conditioned media samples before and after a 6-h in vitro activation of naive CD4+ T lymphocytes with soluble staphylococcal enterotoxin B and anti-CD28. We analyzed samples by ultraperformance liquid chromatography ion mobility-mass spectrometry. We determined the impact of HCA on the CD4+ T lymphocyte exometabolome and identified potential biomarker metabolites by multivariate statistical analyses. We discovered that: 1) CD4+ T lymphocytes exposed to HCA exhibit divergent exometabolomic profiles in both naive and activated states; 2) ∼30% of detected metabolites differentially expressed in response to activation were unique to HCA-positive CD4+ T lymphocytes; 3) metabolic pathways associated with glutathione detoxification and tryptophan degradation were altered in HCA-positive CD4+ T lymphocytes; and 4) flow cytometry and cytokine analyses suggested a bias toward a TH1-biased immune response in HCA-positive samples. HCA exposure primes the neonatal adaptive immune processes by inducing changes to the exometabolomic profile of fetal CD4+ T lymphocytes. These exometabolomic changes may link HCA exposure to TH1 polarization of the neonatal adaptive immune response.

Metabolic phenotyping of mucopolysaccharidoses using untargeted liquid chromatography ion mobility mass spectrometry-based strategy

Metabolic phenotyping of mucopolysaccharidoses using untargeted liquid chromatography ion mobility mass spectrometry-based strategy

Implementation of an untargeted liquid chromatography ion mobility-mass spectrometry-based metabolomics method for inherited metabolic diseases investigation

High-resolution mass spectrometry coupled with pattern recognition techniques is an established tool to perform comprehensive metabolite profiling of biological datasets. Interpreting untargeted metabolomic data requires robust, reproducible and reliable analytical methods to translate results into biologically relevant and actionable knowledge. The analyses of biological samples were developed based on ultra-high performance liquid chromatography (UHPLC) coupled to ion mobility - mass spectrometry (IM-MS). An innovative strategy for optimizing simultaneously the analytical conditions for untargeted UHPLC-IM-MS methods is proposed using an experimental design approach. Optimization experiments were conducted through a screening process designed to identify the influential factors that have significant effects on the selected responses (total number of peaks and number of reliable peaks). For this purpose, full and fractional factorial designs were used while partial least squares regression was used for experimental design modeling and optimization of parameter values. The total number of peaks yielded the best predictive model and is used for the optimization of parameters settings. Fifty-five urine samples have been used to assess the developed method. Twenty-two controles and 33 diseases allocated to five different groups: propionic aciduria, cystinuria, tyrosiniemia, mucopolysaccharidoses (I, IV and VI) and creatine metabolism defect have been metabolically profiled. Multivariate modeling has been performed on the data. This pilot study yielded predictive models that allowed clear classification of the different diseases. These promising results highlight the potential of this new approach in the screening of IEMs and in patient stratification. An assay on a larger cohort is needed to assess the clinical validity of the method. (This work is co-supported by European Union, Region Normandie, Inserm, CNRS, Normandy University and IRIB. Europe gets involved in Normandie with European Regional Development Fund (ERDF).)

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.

Antimicrobial drug resistance affects broad changes in metabolomic phenotype in addition to secondary metabolism

Bacteria develop resistance to many classes of antibiotics vertically, by engendering mutations in genes encoding transcriptional and translational apparatus. These severe adaptations affect global transcription, translation, and the correspondingly affected metabolism. Here, we characterize metabolome scale changes in transcriptional and translational mutants in a genomically characterized Nocardiopsis, a soil-derived actinomycete, in stationary phase. Analysis of ultra-performance liquid chromatography-ion mobility-mass spectrometry metabolomic features from a cohort of streptomycin- and rifampicin-resistant mutants grown in the absence of antibiotics exhibits clear metabolomic speciation, and loadings analysis catalogs a marked change in metabolic phenotype. Consistent with derepression, up to 311 features are observed in antibiotic-resistant mutants that are not detected in their progenitors. Mutants demonstrate changes in primary metabolism, such as modulation of fatty acid composition and the increased production of the osmoprotectant ectoine, in addition to the presence of abundant emergent potential secondary metabolites. Isolation of three of these metabolites followed by structure elucidation demonstrates them to be an unusual polyketide family with a previously uncharacterized xanthene framework resulting from sequential oxidative carbon skeletal rearrangements. Designated as "mutaxanthenes," this family can be correlated to a type II polyketide gene cluster in the producing organism. Taken together, these data suggest that biosynthetic pathway derepression is a general consequence of some antibiotic resistance mutations.

An approach to enhancing coverage of the urinary metabonome using liquid chromatography–ion mobility–mass spectrometry

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC–IM–MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge ( m/ z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 μs) during each IMS separation (∼13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC–MS. Preliminary results indicate that spectral quality is improved when using LC–IM–MS, compared to direct injection IM–MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC–IM–MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).

Developing liquid chromatography ion mobility mass spectometry techniques

When a packet of ions in a buffer gas is exposed to a weak electric field, the ions will separate according to differences in their mobilities through the gas. This separation forms the basis of the analytical method known as ion mobility spectroscopy and is highly efficient, in that it can be carried out in a very short time frame (micro- to milliseconds). Recently, efforts have been made to couple the approach with liquid-phase separations and mass spectrometry in order to create a high-throughput and high-coverage approach for analyzing complex mixtures. This article reviews recent work to develop this approach for proteomics analyses. The instrumentation is described briefly. Several multidimensional data sets obtained upon analyzing complex mixtures are shown in order to illustrate the approach as well as provide a view of the limitations and required future work.