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.
Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.
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
Metabolomics experiments require chemical identifications, often through MS/MS analysis. In mass spectrometry imaging (MSI), this necessitates running several serial tissue sections or using a multiplex data acquisition method. We have previously developed a multiplex MSI method to obtain MS and MS/MS data in a single experiment to acquire more chemical information in less data acquisition time. In this method, each raster step is composed of several spiral steps and each spiral step is used for a separate scan event (e.g., MS or MS/MS). One main limitation of this method is the loss of spatial resolution as the number of spiral steps increases, limiting its applicability for high-spatial resolution MSI. In this work, we demonstrate multiplex MS imaging is possible without sacrificing spatial resolution by the use of overlapping spiral steps, instead of spatially separated spiral steps as used in the previous work. Significant amounts of matrix and analytes are still left after multiple spectral acquisitions, especially with nanoparticle matrices, so that high quality MS and MS/MS data can be obtained on virtually the same tissue spot. This method was then applied to visualize metabolites and acquire their MS/MS spectra in maize leaf cross-sections at 10 μm spatial resolution.
Abstract Two programs, ACEACQ and ACEANN, provide data acquisition and analysis for Size Exclusion Chromatography (SEC). ACEACQ is used for data logging, real-time smoothing and data reduction by boxcar averaging. ACEACQ requires that traditional SEC hardware be coupled with a Hewlett Packard data logger and microcomputer. A Mettler electronic balance is connected to the computer in a novel fashion to continuously monitor the mass of solvent eluted during an experiment. ACEANN provides analysis for the data collected by ACEACQ. Calculations include molecular weight averages, molecular hydrodynamic diameter averages and column separation efficiency parameters. Significant reductions in analysis time have been realized along with improvements in accuracy of data acquisition, analysis and experimental reproducibility. 相似文献
Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide
(CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry
methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss
(NL) scans-dependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry.
GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound.
Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey
scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition
data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts,
respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular
weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed
from rat liver microsomes. In addition to known GSH- and CN-trapped reactive metabolites, several new CN adducts of nefazodone
were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive
metabolites and can be used as a high-throughput method in drug discovery. 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
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
The objective of the present study was to investigate mesocarb metabolism in humans. Samples obtained after administration
of mesocarb to healthy volunteers were studied. The samples were extracted at alkaline pH using ethyl acetate and salting-out
effect to recover metabolites excreted free and conjugated with sulfate. A complementary procedure was applied to recover
conjugates with glucuronic acid or with sulfate consisting of the extraction of the urines with XAD-2 columns previously conditioned
with methanol and deionized water; the columns were then washed with water and finally eluted with methanol. In both cases,
the dried extracts were reconstituted and analyzed by ultra-performance liquid chromatography–tandem mass spectrometry. Chromatographic
separation was carried out using a C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size) and a mobile phase consisting of water and acetonitrile with 0.01% formic
acid with gradient elution. The chromatographic system was coupled to a mass spectrometer with an electrospray ionization
source working in positive mode. Metabolic experiments were performed in multiple-reaction monitoring mode by monitoring one
transition for each potential mesocarb metabolite. Mesocarb and 19 metabolites were identified in human urine, including mono-,
di-, and trihydroxylated metabolites excreted free as well as conjugated with sulfate or glucuronic acid. All metabolites
were detected up to 48 h after administration. The structures of most metabolites were proposed based on data from reference
standards available and molecular mass and product ion mass spectra of the peaks detected. The direct detection of mesocarb
metabolites conjugated with sulfate and glucuronic acid without previous hydrolysis has been described for the first time.
Finally, a screening method to detect the administration of mesocarb in routine antidoping control analyses was proposed and
validated based on the detection of the main mesocarb metabolites in human urine (p-hydroxymesocarb and p-hydroxymesocarb sulfate). After analysis of several blank urines, the method demonstrated to be specific. Extraction recoveries
of 100.3 ± 0.8 and 105.9 ± 10.8 (n = 4), and limits of detection of 0.5 and 0.1 ng mL−1 were obtained for p-hydroxymesocarb sulfate and p-hydroxymesocarb, respectively. The intra- and inter-assay precisions were estimated at two concentration levels, 50 and 250 ng
mL−1, and relative standard deviations were lower than 15% in all cases (n = 4). 相似文献
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
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 paper describes a method for determination of 27 mycotoxins and other secondary metabolites in maize silage. The method
focuses on analytes which are known to be produced by common maize and maize-silage contaminants. A simple pH-buffered sample
extraction was developed on the basis of a very fast and simple method for analysis of multiple pesticide residues in food
known as QuEChERS. The buffering effectively ensured a stable pH in samples of both well-ensiled maize (pH < 4) and of hot
spots with fungal infection (pH > 7). No further clean-up was performed before analysis using liquid chromatography–tandem
mass spectrometry. The method was successfully validated for determination of eight analytes qualitatively and 19 quantitatively.
Matrix-matched calibration standards were used giving recoveries ranging from 37% to 201% with the majority between 60% and
115%. Repeatability (5–27% RSDr) and intra-laboratory reproducibility (7–35% RSDIR) was determined. The limit of detection (LOD) for the quantitatively validated analytes ranged from 1 to 739 μg kg−1. Validation results for citrinin, fumonisin B1 and fumonisin B2 were unsatisfying. The method was applied to 20 selected silage samples and alternariol monomethyl ether, andrastin A, alternariol,
citreoisocoumarin, deoxynivalenol, enniatin B, fumigaclavine A, gliotoxin, marcfortine A and B, mycophenolic acid, nivalenol,
roquefortine A and C and zearalenone were detected. 相似文献
