Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for characterizing multiple samples of complex mixtures with similar compositions. This article addresses a data acquisition strategy for collecting a maximal number of unique, high-quality MS/MS during LC-MS/MS analysis of multiple samples. Based on the concept that a component only needs to be identified once when analyzing multiple samples with similar compositions, an automated intersample data-dependent acquisition strategy was developed. The strategy is based on precursor ion exclusion (PIE) and is implemented in MassAnalyzer in an automated fashion for Thermo Scientific (San Jose, CA, USA) mass spectrometers. In this method, MassAnalyzer submits one sample at a time to the sample queue. After data acquisition of each sample, MassAnalyzer automatically analyzes the data to generate a PIE list based on the MS/MS precursor ions, merges this list with the list generated from previous runs, adds the list to the MS method file, and submits the next sample to the queue. The PIE list contains both m/z value and time window for each precursor ion, and is generated intelligently so that if an MS/MS is insufficient for identifying the peak of interest, it will be collected again near the top of the peak in the next run. Therefore, the strategy maximizes both quality and the number of unique MS/MS. When automated PIE was used to acquire LC-MS/MS data of an antibody tryptic digest and a soy hydrolysate sample, the number of identified ions increased by 52% and 93%, respectively, compared with data acquired without using PIE. 相似文献
Untargeted omics analyses aim to comprehensively characterize biomolecules within a biological system. Changes in the presence or quantity of these biomolecules can indicate important biological perturbations, such as those caused by disease. With current technological advancements, the entire genome can now be sequenced; however, in the burgeoning fields of lipidomics, only a subset of lipids can be identified. The recent emergence of high resolution tandem mass spectrometry (HR-MS/MS), in combination with ultra-high performance liquid chromatography, has resulted in an increased coverage of the lipidome. Nevertheless, identifications from MS/MS are generally limited by the number of precursors that can be selected for fragmentation during chromatographic elution. Therefore, we developed the software IE-Omics to automate iterative exclusion (IE), where selected precursors using data-dependent topN analyses are excluded in sequential injections. In each sequential injection, unique precursors are fragmented until HR-MS/MS spectra of all ions above a user-defined intensity threshold are acquired. IE-Omics was applied to lipidomic analyses in Red Cross plasma and substantia nigra tissue. Coverage of the lipidome was drastically improved using IE. When applying IE-Omics to Red Cross plasma and substantia nigra lipid extracts in positive ion mode, 69% and 40% more molecular identifications were obtained, respectively. In addition, applying IE-Omics to a lipidomics workflow increased the coverage of trace species, including odd-chained and short-chained diacylglycerides and oxidized lipid species. By increasing the coverage of the lipidome, applying IE to a lipidomics workflow increases the probability of finding biomarkers and provides additional information for determining etiology of disease.
Eicosanoids are potent lipid mediators involved in numerous physiological and pathophysiological processes. Precursors are polyunsaturated fatty acids liberated from membrane phospholipids. Thus, profiling and quantification of these molecules has gained a lot of attention during last years. Eicosanoids and phospholipids are commonly profiled by LC-MS/MSbecause this technique allows accurate quantification within acceptable run-times. This article therefore focuses on liquid chromatography and the ESI-MS/MS analysis of proinflammatory lipid mediators, particularly arachidonic acid (C20:4) derived eicosanoids and their precursors phospholipids. Recent analytical developments for quantification of these compounds are highlighted and analytical challenges are discussed. Furthermore, applications such as the use of these molecules as biomarkers are presented. 相似文献
Deer antler is a globally widely used precious natural medicine and the material of deer horn gelatin. However, identification of deer antler species based on traditional approaches are problematic because of their similarity in appearance and physical-chemical properties. In this study, we performed a comprehensive antler peptidome analysis using a label-free approach: nano LC-Orbitrap MS was applied to discover peptide biomarkers in deer adult beta-globin (HBBA), and HPLC-Triple Quadrupole MS was used to verify their specificity. Nineteen peptide biomarkers were found, on which foundation a strategy for antlers and a strategy for antler mixtures such as flakes or powder are provided to identify seven species of deer antler including Eurasian elk (Alces alces), reindeer (Rangifer tarandus), white-tailed deer (Odocoileus viginianus), white-lipped deer (Przewalskium albirostris), fallow deer (Dama dama), sika deer (Cervus nippon), and red deer (Cervus elaphus) simultaneously. It is worth noting that our search found that the HBBA gene of sika deer, red deer, and North American wapiti (Cervus canadensis) in China may have undergone severe genetic drifts. 相似文献
Liquid chromatography coupled to tandem mass spectrometry has been compared to shotgun analysis with the objective of finding the best compromise for a single run analysis of whole cell phospholipids. Hydrophilic interaction liquid chromatography (HILIC), normal phase (NP), and reversed phase (RP) liquid chromatography were evaluated with reference phospholipids belonging to phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) classes. NP-HPLC- and RP-HPLC-ESI-MS/MS were applied to yeast phospholipidome analysis, using a wild-type strain and two strains defective for acyltransferases that are known to be involved in de novo phospholipid synthesis or phospholipid remodeling. The MRM mode was used for relative quantitation of individual compounds based on reference phospholipids bearing fatty acid chains with an odd number of carbon atoms. Combined LC-MS/MS was found superior to shotgun analysis, leading to a larger number of quantified species than shotgun analysis. Finally, RP-HPLC-MS/MS was the preferred method for its higher selectivity, robustness, and better repeatability. 相似文献
Carbonyl-containing metabolites widely exist in biological samples and have important physiological functions. Thus, accurate and sensitive quantitative analysis of carbonyl-containing metabolites is crucial to provide insight into metabolic pathways as well as disease mechanisms. Although reversed phase liquid chromatography electrospray ionization mass spectrometry (RPLC-ESI-MS) is widely used due to the powerful separation capability of RPLC and high specificity and sensitivity of MS, but it is often challenging to directly analyze carbonyl-containing metabolites using RPLC-ESI-MS due to the poor ionization efficiency of neutral carbonyl groups in ESI. Modification of carbonyl-containing metabolites by a chemical derivatization strategy can overcome the obstacle of sensitivity; however, it is insufficient to achieve accurate quantification due to instrument drift and matrix effects. The emergence of stable isotope-coded derivatization (ICD) provides a good solution to the problems encountered above. Thus, LC-MS methods that utilize ICD have been applied in metabolomics including quantitative targeted analysis and untargeted profiling analysis. In addition, ICD makes multiplex or multichannel submetabolome analysis possible, which not only reduces instrument running time but also avoids the variation of MS response. In this review, representative derivatization reagents and typical applications in absolute quantification and submetabolome profiling are discussed to highlight the superiority of the ICD strategy for detection of carbonyl-containing metabolites. 相似文献
A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D2O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H2O or D2O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site(s) of modification for each metabolite. 相似文献
A LC-MS/MS method with enhanced sensitivity and specificity was established for monitoring microcystin-LR (MC-LR) in drinking water supplies in southern Taiwan. The enhanced sensitivity was achieved by the selection of a doubly charged MC-LR as the precursor ion to result in an multiple reaction monitoring (MRM) pair ions of m/z 498.6 --> 135.0. Using this ion pair, a record low detection limit of 2 pg was achieved on column, found in the available literature. A sample preparation method involving C8 solid-phase extraction gave satisfactory recoveries of the analyte. Nodularin, with structural similarity to MC-LR, was used as an internal standard to minimize matrix effects of water samples collected from six different water reservoirs in southern Taiwan, where MC-LR was detected at sub-ppb levels in all the reservoirs. The best precision and accuracy of this method were found with samples prepared to contain MC-LR at 0.1 and 1 microg l(-1). This new method requires considerably smaller water sample volumes because of enhanced quantification sensitivity and hence reduces the time needed for analysis. It should serve as a useful example for method development for monitoring other members of the microcystin family in drinking water supplies. 相似文献
This work describes the coupling of the IR-MALDESI imaging source with the Q Exactive mass spectrometer. IR-MALDESI MSI was used to elucidate the spatial distribution of several HIV drugs in cervical tissues that had been incubated in either a low or high concentration. Serial sections of those analyzed by IR-MALDESI MSI were homogenized and analyzed by LC-MS/MS to quantify the amount of each drug present in the tissue. By comparing the two techniques, an agreement between the average intensities from the imaging experiment and the absolute quantities for each drug was observed. This correlation between these two techniques serves as a prerequisite to quantitative IR-MALDESI MSI. In addition, a targeted MS2 imaging experiment was also conducted to demonstrate the capabilities of the Q Exactive and to highlight the added selectivity that can be obtained with SRM or MRM imaging experiments. Fig. a
Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C18 column (3.0?×?50 mm, 2.7 μm) with a mobile phase consisting of 0.1 % formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271?→?159 (estrone), and m/z 361?→?163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.
Figure
Determination of estrone formation by LC-MS/MS analysis for aromatase inhibitor screening 相似文献
Mass spectrometry (MS) is widely used for the identification of chemical compounds by matching the experimentally acquired mass spectrum against a database of reference spectra. However, this approach suffers from a limited coverage of the existing databases causing a failure in the identification of a compound not present in the database. Among the computational approaches for mining metabolite structures based on MS data, one option is to predict molecular fingerprints from the mass spectra by means of chemometric strategies and then use them to screen compound libraries. This can be carried out by calibrating multi-task artificial neural networks from large datasets of mass spectra, used as inputs, and molecular fingerprints as outputs. In this study, we prepared a large LC-MS/MS dataset from an on-line open repository. These data were used to train and evaluate deep-learning-based approaches to predict molecular fingerprints and retrieve the structure of unknown compounds from their LC-MS/MS spectra. Effects of data sparseness and the impact of different strategies of data curing and dimensionality reduction on the output accuracy have been evaluated. Moreover, extensive diagnostics have been carried out to evaluate modelling advantages and drawbacks as a function of the explored chemical space. 相似文献
We report here a novel approach for the extraction and analysis of thyroid hormones (TH) and their metabolites (THM) from human serum samples. Our method features a compact, 96-well micro-titre plate-based pre-analytic extraction/clean-up workflow combined with an isotope dilution LC-MS/MS-MS3 analytical method. In particular, these features make possible the detection of iodothyronines at their endogenous concentrations in serum differing by a factor of ca. 104, with potential to semi-automate the pre-analytics. The method was validated by the assessment of linearity, lower limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves were linear in the concentration range in sample matrix from 0.1–250 nM for T3, rT3, T4 and 3-T1AM and from 0.005–1 nM for 3,5-T2 and 3,3′-T2. Using a 200-μL sample volume, the analyte dependant LLOQ were in the range 0.005 (3,5-T2) to 0.25 (T4) nM and LLOD were between 0.002 (3,5-T2) and 0.052 nM (T4). We applied the LC-MS/MS-MS3 method to the analysis of a cross section of patients with disorders of the thyroid hormone axis. T4, T3 and rT3 concentrations (± standard deviation) were 120 ± 18, 1.9 ± 0.4 and 0.45 ± 0.09 nM respectively. 3,3′-T2 concentrations (± standard deviation) were 0.079 ± 0.022 nM; 3,5-T2 concentrations were below the LLOQ and/or LLOD in all but a single sample (0.013 nM). This method expands the analytical spectrum to endogenous thyroid hormone metabolites such as 3,5-T2 which exert biological actions and rT3 which may act as surrogate markers for disturbed thyroid hormone metabolism.
