Isotope Cluster-Based Compound Matching in Gas Chromatography/Mass Spectrometry for Non-Targeted Metabolomics

Gas chromatography coupled to mass spectrometry (GC/MS) has emerged as a powerful tool in metabolomics studies. A major bottleneck in current data analysis of GC/MS-based metabolomics studies is compound matching and identification, as current methods generate high rates of false positive and false -negative identifications. This is especially true for data sets containing a high amount of noise. In this work, a novel spectral similarity measure based on the specific fragmentation patterns of electron impact mass spectra is proposed. An important aspect of these algorithmic methods is the handling of noisy data. The performance of the proposed method compared to the dot product, the current gold standard, was evaluated on a complex biological data set. The analysis results showed significant improvements of the proposed method in compound matching and chromatogram alignment compared to the dot product

Table_1_Erythritol synthesis is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway in A549 cells.DOCX

BackgroundErythritol is a predictive biomarker of cardiometabolic diseases and is produced from glucose metabolism through the pentose phosphate pathway (PPP). Little is known regarding the regulation of endogenous erythritol synthesis in humans.ObjectiveIn the present study, we investigated the stimuli that promote erythritol synthesis in human lung carcinoma cells and characterized potential points of regulation along the PPP.MethodsHuman A549 lung carcinoma cells were chosen for their known ability to synthesize erythritol. A549 cells were treated with potential substrates for erythritol production, including glucose, fructose, and glycerol. Using siRNA knockdown, we assessed the necessity of enzymes G6PD, TKT, TALDO, and SORD for erythritol synthesis. We also used position-specific 13C-glucose tracers to determine whether the carbons for erythritol synthesis are derived directly from glycolysis or through the oxidative PPP. Finally, we assessed if erythritol synthesis responds to oxidative stress using chemical and genetic models.ResultsIntracellular erythritol was directly associated with media glucose concentration. In addition, siRNA knockdown of TKT or SORD inhibited erythritol synthesis, whereas siG6PD did not. Both chemically induced oxidative stress and constitutive activation of the antioxidant response transcription factor NRF2 elevated intracellular erythritol.ConclusionOur findings indicate that in A549 cells, erythritol synthesis is proportional to flux through the PPP and is regulated by non-oxidative PPP enzymes.</p

Additional file 1 of Bridging the gap between non-targeted stable isotope labeling and metabolic flux analysis

Supplemental information. List of isotopically enriched compounds and their mass isotopomer distributions; selected mass spectra. (PDF 308 kb

Fragment Formula Calculator (FFC): Determination of Chemical Formulas for Fragment Ions in Mass Spectrometric Data

The accurate determination of mass isotopomer distributions (MID) is of great significance for stable isotope-labeling experiments. Most commonly, MIDs are derived from gas chromatography/electron ionization mass spectrometry (GC/EI-MS) measurements. The analysis of fragment ions formed during EI, which contain only specific parts of the original molecule can provide valuable information on the positional distribution of the label. The chemical formula of a fragment ion is usually applied to derive the correction matrix for accurate MID calculation. Hence, the correct assignment of chemical formulas to fragment ions is of crucial importance for correct MIDs. Moreover, the positional distribution of stable isotopes within a fragment ion is of high interest for stable isotope-assisted metabolomics techniques. For example, ¹³C-metabolic flux analyses (¹³C-MFA) are dependent on the exact knowledge of the number and position of retained carbon atoms of the unfragmented molecule. Fragment ions containing different carbon atoms are of special interest, since they can carry different flux information. However, the process of mass spectral fragmentation is complex, and identifying the substructures and chemical formulas for these fragment ions is nontrivial. For that reason, we developed an algorithm, based on a systematic bond cleavage, to determine chemical formulas and retained atoms for EI derived fragment ions. Here, we present the fragment formula calculator (FFC) algorithm that can calculate chemical formulas for fragment ions where the chemical bonding (e.g., Lewis structures) of the intact molecule is known. The proposed algorithm is able to cope with general molecular rearrangement reactions occurring during EI in GC/MS measurements. The FFC algorithm is able to integrate stable isotope labeling experiments into the analysis and can automatically exclude candidate formulas that do not fit the observed labeling patterns. We applied the FFC algorithm to create a fragment ion repository that contains the chemical formulas and retained carbon atoms of a wide range of trimethylsilyl and <i>tert</i>-butyldimethylsilyl derivatized compounds. In total, we report the chemical formulas and backbone carbon compositions for 160 fragment ions of 43 alkylsilyl-derivatives of primary metabolites. Finally, we implemented the FFC algorithm in an easy-to-use graphical user interface and made it publicly available at http://www.ffc.lu

Methodological detection solution scaffolds response to mechanical stress, depending on the degree of hydration

Title: Methodological detection solution scaffolds response to mechanical stress, depending on the degree of hydration Objectives: Determining the extent of lateral deformation u scaffolds made of PVA polymer electrospinning technique. Identify the extent of differences in transverse deformation for different groups of nanofiber scaffolds made of PVA polymer electrospinning technique. Methods: Research scaffolds, we used a measuring device μ-tester, which has two jaws. For the measurement, we chose uniaxial tension test in -tester and record the fluorescence microscope was used with HD camera Olympus 320 for online video recording. Results: The results of this study showed that the ratio of the samples U: L and crosslinking time affects the degree of lateral deformation of the samples scaffolds. Samples scaffolds are compressible, some groups even reached the limits of incompressibility 0.5 Poisson's ratio. Keywords: Poisson, Poisson's ratio, scaffold, nanofiber scaffold, scaffold hydrated, electrospun scaffold, lateral deformatio

Additional file 5 of The role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells

Table S3. Mass isotopomer distributions (MIDs) from [13C5]glutamine in PH5CH8 immortalized human hepatocytes treated for 36 h with 50 ng/mL OSM or left untreated. MIDs were corrected for natural isotope abundance. (PDF 94.2 kb

S2 Fig -

(A) Differentiated caspase-4-deficient BLaER1 cells were stimulated as in Fig 3A. IL-1β was assessed from the harvested supernatants. (B) LPS-primed differentiated WT BLaER1 cells were preincubated with TAK242 (2 μM, 30 min) then activated with TcdB (20 ng/ml) or nigericin (8 μM) for 2 h. Harvested supernatant was assessed for IL-1β. (C) Differentiated WT BLaER1 cells were preincubated with TAK242 then stimulated with LPS for 4 h. TNFα was assessed from the supernatant. (D) TNFα was measured for THP-1 cells from Fig 3G. Mean and SEM shown for 3 independent experiments. (E) LPS-primed (10 ng/ml, 3 h) human macrophages were treated either TcdB or the TcdB NXN mutant lacking glucosyltrasferase activity (20 ng/ml, 2.5 h). Supernatant was harvested and assessed for IL-1β or TNFα. For (A-C), the mean and SD of 3 technical replicates shown, representative of 3 independent experiments. For (D) and (E), the mean and SEM shown for 3 independent experiments. * p S1 Data) in the tab S2B–S2E Fig. (PDF)</p

The raw data underlying the graphs shown in Figs 1–7 and S1–S3.

Each tab contains the data from one figure, the data is labeled as in the graphs shown in the main figure. (XLSX)</p

S1 Fig -

(A) Immunoblot of Rac glucosylation status in either monocytes or hMDM following treatment with the listed toxins (NXN variants lack glucosyltransferase activity). Representative of 3 experiments. (B) Immunoblot of hMDM lysate sequentially probed with the α-Pyrin antibody preabsorbed against HEKs transfected with Pyrin, then with the α-Pyrin antibody preabsorbed against those transfected then empty vector (control). (C) Pyrin expression in monocytes or hMDM from 3 different donors. (D) Actin staining following incubation of monocytes or macrophages with or without LPS and TcdB. Treated cells were fixed and stained with Phalloidin 647 to detect actin (red) or with DAPI to detect nuclei (blue). White arrows highlight the changes in actin distribution between the 2 conditions. Images are representative from 3 separate donors. (E) IL-1β release from LPS-primed hMDM differentiated for 7 days in M-CSF and stimulated with either TcdA or TcdB +/ CP-456,773. Mean and SEM shown for 3 independent donors, * p S1 Data) in the tab S1E Fig. (PDF)</p

Complete immunoblots for Figs 1–7 and S1–S3.

The excerpted portion of the immunoblot shown in the relevant figure is highlighted by a black box. (PDF)</p