liquid chromatography mass spectrometry  (SCIEX)

Journal: Scientific Reports

Article Title: Cross-Platform Comparison of Untargeted and Targeted Lipidomics Approaches on Aging Mouse Plasma

doi: 10.1038/s41598-018-35807-4

Figure Lengend Snippet: Qualitative and quantitative cross-platform comparison. ( A ) Schematic illustration of the workflows for both untargeted LC-MS and targeted Lipidyzer platforms. Lipids were extracted from mouse plasma using a modified Folch method. Two internal standards (IS) were spiked-in prior to lipid extraction and were used for normalization in the LC-MS approach. The Lipidyzer platform requires the addition of a mixture of IS that is used for quantification, which in this case was added to the lipid extracts prior to analysis. Processing of LC-MS data consisted in six steps: (1) peak extraction, (2) retention time alignment, (3) quantification, (4) normalization, (5) identification, and (6) manual validation. In contrast, the Lipidyzer platform quantifies a pre-determined list of lipids, a process that is automated by the software provided with the platform. While our untargeted LC-MS provides relative lipid abundances, the Lipidyzer platform gives an accurate quantification in nmol/g (estimated concentration) based on the IS. DMS: Differential Mobility Spectrometry, MRM: Multiple Reaction Monitoring. ( B ) Venn diagram representing lipid coverage of each approach and their overlap. Note that the lipids detected with the LC-MS approach were converted to match the level of information provided by the Lipidyzer platform. Lipid coverage assessment was performed using data from three injections of a pool sample. ( C ) Barplot depicting the overlap in coverage by lipid class. ( D ) Boxplot representing intra- and inter-day precisions with the indicated platform. Precisions were calculated on IS spiked in a plasma matrix using five replicate injections on the same day (intra-day) and on three consecutive days (inter-day). Lipidomics Workflow Manager (LWM) recommended concentrations spanning 3 orders of magnitude were used to determine precisions. ( E ) Boxplot depicting accuracies of both platforms using IS at LWM recommended concentrations. Accuracy was calculated on each IS using a 7-point dilution series. ( F ) Pearson correlation coefficient of lipids identified by both platforms across all biological samples (87 lipids). TAG were excluded from the analysis because of nomenclature discrepancies between the two approaches. ( G ) Scatterplots showing examples of one PC and one PE with a Pearson correlation coefficient close to the median of the corresponding lipid class. TAG: triacylglycerol, DAG: diacylglycerol, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, LPC: lysophosphatidylcholine, LPE: lysophoshatidylethanolamine, SM: sphingomyelin, CER: ceramide, CE: cholesterol ester, FFA: free fatty acid, MISC: miscellaneous.

Article Snippet: These questions prompted us to compare a conventional untargeted LC-MS approach with the targeted Lipidyzer platform.

Techniques: Liquid Chromatography with Mass Spectroscopy, Modification, Software, Concentration Assay

Journal: Toxins

Article Title: A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

doi: 10.3390/toxins7104216

Figure Lengend Snippet: Correlation of results obtained by both MNP-bsELISA and LC-MS/MS for zearalenone detection in natural cereal and feed samples.

Article Snippet: Quantitative LC-MS/MS results were analyzed using Analyst software (AB SCIEX, Framingham, MA, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: PLoS ONE

Article Title: Rv2607 from Mycobacterium tuberculosis Is a Pyridoxine 5?-Phosphate Oxidase with Unusual Substrate Specificity

doi: 10.1371/journal.pone.0027643

Figure Lengend Snippet: PLP formation catalyzed by PNPOx and associated vitamin B6 metabolic pathways. The role of Rv2607 is shown in red. In E. coli , PNPOx catalyzes the last step in the DXP-dependent PLP biosynthetic pathway [2] . Most organisms capable of PLP biosynthesis produce PLP via PLP synthase, a macromolecular complex consisting of Pdx1 and Pdx2 [2] . Organisms with genes that encode both PLP synthase and PNPOx likely use PNPOx to salavage PLP from PNP and PMP, which are produced by enzymes that use PLP as a cofactor.

Article Snippet: Extracted cofactor from Rv2607 and authentic FMN were analyzed with a Q Star Elite Mass Spectrometer (Applied Biosystems/MDS Sciex) by electrospray ionization positive ion mode with a gold-plated nanospray tip ( ).

Techniques: Plasmid Purification, Produced

Journal: PLoS ONE

Article Title: Rv2607 from Mycobacterium tuberculosis Is a Pyridoxine 5?-Phosphate Oxidase with Unusual Substrate Specificity

doi: 10.1371/journal.pone.0027643

Figure Lengend Snippet: Nano-ESI mass spectrum of intact Rv2607 with co-purified FMN. The major species, C , has peaks with charges ranging from 15+ to 12+ in the 3500-4500 m/z range that correspond to a molecular weight of 55376±12. This experimental mass is consistent with the calculated mass for the complex of dimeric Rv2607 with one molecule of FMN bound (55442 Da), which is derived from the amino acid sequence of the 6x-histidine tagged Rv2607 (54986 Da; 27493 Da per protomer) and the molecular weight of FMN (456 Da). Monomeric tagged Rv2607 and truncated Rv2607 monomer (missing 10-11 residues), are present in solution and correspond to molecular weights of A (27450±52 Da) and B (26215±54 Da) respectively. A minor dimeric species comprised of full length and truncated monomer is represented as species D . Higher molecular weight oligomers (trimers E and tetramers F with molecular weights of 110.6 and 82.7 kDa, respectively) are electrospray-induced non-specific association of monomers and dimers.

Article Snippet: Extracted cofactor from Rv2607 and authentic FMN were analyzed with a Q Star Elite Mass Spectrometer (Applied Biosystems/MDS Sciex) by electrospray ionization positive ion mode with a gold-plated nanospray tip ( ).

Techniques: Purification, Molecular Weight, Derivative Assay, Sequencing

Journal: PLoS ONE

Article Title: Rv2607 from Mycobacterium tuberculosis Is a Pyridoxine 5?-Phosphate Oxidase with Unusual Substrate Specificity

doi: 10.1371/journal.pone.0027643

Figure Lengend Snippet: Reverse-phase HPLC, NMR, and spectrophotometric analysis of the PNPOx activity of Rv2607. (A) HPLC chromatograms (270 nm) of reaction mixtures containing PNP (i-iv) or PMP (v-viii), FMN, and Rv2607, and the associated control reactions. All reactions were carried out in 25 mM potassium phosphate buffer (pH 7.8), incubated at 25°C for 3 h, and quenched with DNPH (0.7 mM final concentration). Where present, the reaction components were at the following concentrations: 1 mM PNP or PMP, 10 µM FMN, and 10 µM Rv2607. Reaction mixtures contained: (i) PNP, FMN, and Rv2607, (ii) PNP and FMN (enzyme negative control), (iii) PNP and Rv2607 (no added FMN), (iv) FMN and Rv2607 (substrate negative control), (v) PMP, FMN, and Rv2607, (vi) PMP and FMN (enzyme negative control), (vii) PMP and Rv2607 (no added FMN), (viii) FMN and Rv2607 (substrate negative control). The peak to the right of PLP-DNPH is DNPH, which has a retention time of 8.6 min. (B) 1 H NMR analysis of the conversion of PNP into PLP with time. The enzymatic reaction (1 mM PNP, 28 µM enzyme, 10% D 2 O in 25 mM potassium phosphate buffer, pH 7.8) was incubated for 18 hours at 298 K in the spectrometer. Left; stacked 1 H NMR spectra recorded at various time points and after the addition of authentic PLP. The starred peak corresponds to PLP hydrate. Right; a plot of percent substrate conversion versus time. Substrate conversion was determined by comparing integrals of the C2- 1 H signals associated with PLP (7.80 ppm) to that of PNP (7.75 ppm). (C) Michaelis-Menten plot for Rv2607 with PNP as a substrate. The rate of PLP formation was monitored spectrophotometrically (λ max = 388 nm, ε = 4900 cm −1 M −1 ) for various concentrations of PNP. All solutions were made in 100 mM potassium phosphate buffer (pH 7.8).

Article Snippet: Extracted cofactor from Rv2607 and authentic FMN were analyzed with a Q Star Elite Mass Spectrometer (Applied Biosystems/MDS Sciex) by electrospray ionization positive ion mode with a gold-plated nanospray tip ( ).

Techniques: High Performance Liquid Chromatography, Nuclear Magnetic Resonance, Activity Assay, Incubation, Concentration Assay, Negative Control, Plasmid Purification

Journal: bioRxiv

Article Title: Host cholesterol dependent activation of VapC12 toxin enriches persister population during Mycobacterium tuberculosis infection

doi: 10.1101/856286

Figure Lengend Snippet: VapC12 ribonuclease toxin targeting proT is essential for cholesterol-mediated growth regulation in Mtb A) Diagrammatic representation of toxin–antitoxin vapBC12 locus. B) Relative expression of proT tRNA through qRTPCR in the vapC12 mutant relative to the wild-type H37Rv strain grown in media containing glycerol, cholesterol, and palmitate as the sole carbon source. C) Growth curve of M. bovis BCG strain expressing vapC12 , vapB12 , and vapBC12 in the pUV15-tetO expression system under the tet-inducible promoter in 7H9+OADC media. Anhydrotetracycline (ATc), an inducer of the tet operon, was used at a concentration of 100 ng/mL and replenished every fourth day. D) Two-fold serial dilutions (N/2, N/4, N/8, N/16, N/32) of the log phase growing culture BCG:pUV15 tetO:vapC12 strain grown in 7H9 broth were spotted on 7H11 agar plates with or without ATc. E) Relative quantification of the transcript levels of proT gene in BCG:pUV15-tetO:vapC12 grown in 7H9 media with or without ATc by qRT-PCR. F) RNase activity of purified wild-type and mutant VapC12 toxins against in vitro transcribed tRNA substrates. Different wells of the gel denote different combination of tRNA transcript and purified proteins viz; (A) Wild-type VapC12 toxin protein incubated with proT, (B) proT tRNA only with no protein, (C) wild-type VapC12 toxin protein incubated with proU, (D) proU tRNA only, (E) mutant VapC12D 94 A toxin protein incubated with proT, and (F) mutant VapC12D 5 A toxin protein incubated with proT. Each reaction was incubated at 37°C for 3 hours. The products of each of the reaction were run on a 3% agarose gel and visualized by adding ethidium bromide followed by exposure to UV light. G) Relative density of marked RNA bands in Fig 2F quantified using ImageJ. The experiment (2F) was repeated three times, and data plotted represent the mean ± SEM. H) Schematic representation of the protocol for the experiment to demonstrate cholesterol-specific dissociation of the antitoxin. I) Western blot for cholesterol-specific dissociation and degradation of the antitoxin from the toxin–antitoxin complex. The His-tagged antitoxin was tracked using an anti-His antibody in the cell lysate of BCG overexpressing His-tagged antitoxin as a part of the toxin–antitoxin complex. Cell lysates were prepared by sampling cultures grown in both glycerol and cholesterol media at different time points and probed with an anti-His antibody. J) Western blot of the protein lysates prepared from BCG overexpressing toxin-antitoxin locus (VapBC12) with His tagged antitoxin VapB12 and BCG strain with N-terminal His tagged VapBC12 where in lysine residue of AT is converted to alanine (VapB K:A C12). Immunoprecipitation was performed using mouse anti-His antibody and probed with rabbit anti-acetyl lysine and anti-His antibodies. To normalize for the amount of the protein, three-fold higher concentration of protein was loaded in the cholesterol-grown BCG sample. K) Mass spectrometry analysis of His-tagged antitoxin protein isolated from BCG overexpressing VapBC12 complex grown in glycerol and cholesterol media. Tryptic digest of immunoprecipitated samples from glycerol and cholesterol grown cultures were analysed by LC-MS/MS (Sciex Triple TOF 5600). Representative MS/MS spectrum of peptide from glycerol grown sample, ELLHELK(Ac)AR was acetylated and displays mass shift corresponding acetylation (m/z 416.26) when compared to the unmodified peptide from cholesterol grown sample. L) Growth curve of BCG overexpressing toxin–antitoxin ( vapBC12 ) and lysine mutant ( vapB K:A C12 ) in a minimal media containing 0.1% glycerol as the carbon source. Bacterial enumeration was performed at day 7 after inoculation by CFU plating on 7H11+OADC plates. Experiment was performed in triplicates, and data plotted represent the mean ± SEM. Data were analysed using unpaired Student’s t test. *P

Article Snippet: Mass spectrometry data analysis All raw mass spectrometry files were searched in Protein Pilot software v. 5.0.1 (SCIEX) with the Paragon algorithm.

Techniques: Expressing, Mutagenesis, Concentration Assay, Quantitative RT-PCR, Activity Assay, Purification, In Vitro, Incubation, Agarose Gel Electrophoresis, Western Blot, Sampling, Immunoprecipitation, Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy