Quanlynx

The methionine-associated metabolites were determined from wild-type and Dadi1 homozygous mutant ovaries raised under the restricted diet condition. Twenty ovaries per genotype were collected and homogenized in 80% MeOH. The extraction samples were centrifuged at 12000 rpm for 10 min at 4°C. The supernatants were transferred into clean tubes, dried with N2, and stored at −80°C until UPLC/MS analysis. Samples were subjected to Ultra performance liquid chromatography coupled with triple quadrupole massspectrometry (UPLC/TQMS) system. Results were further analyzed using the Masslynx™ 4.0 and QuanLynx™ (Waters) software systems. The UPLC/TQMS analysis was conducted in the Metabolomics Core Laboratory of the Healthy Aging Research Center, Chang Gung University.

A graphical overview of the data processing steps is given in Fig. 1. In short, automatically integrated LC-MS data (MarkerLynx, Waters Inc) was cleaned from non-endogenous, background noise signals. The remaining signals were corrected for drift if detected in QC samples. Next, the signals were normalized with the median fold change method^{47 (link), 48 (link)}. Adjusted data were subjected to null hypothesis significance testing by either two-sample t-test or Wilcoxon signed rank test, depending on the outcome of the Shapiro-Wilk test for normality. Also taken in account was the homogeneity of variance via the Barlett test. Data adjustments and statistical analysis were done in R (R 3.2.3, R Core Team, 2015).
After this first selection, rough identifications based on exact mass to charge ratios (m/z), isotope distributions, fragmentation patterns and database hits (HMDB, METLIN) were performed. Peaks in the extracted ion-chromatograms of features with a putative identification were manually reintegrated (QuanLynx, Waters Inc.) and the new values were again subjected to data adjustments and significance testing. The metabolite markers from this second selection were used for pathway enrichment. The enriched feature list was subjected to a final identification step with chemical standards.

Plasma drug concentrations were measured using liquid chromatography coupled with tandem mass spectrometry (ACQUITY UPLC-Quattro Premier XE system, Waters, Milford, MA, USA) in a device equipped with an ACQUITY UPLC BEH shield (RP18, 1.7 μm) and a 2.1×100 mm column (Waters). All the data were acquired and processed using MassLynx software (version 4.1) with QuanLynx (Waters). Positive-ion electrospray tandem mass spectroscopy, operated under the multiple reaction monitoring mode, was used to detect mass transitions (parent to daughter ion), with m/z 399.48 to 283.1 for sunitinib, m/z 371.27 to 283.1 for N-desethylsunitinib, m/z 465.35 to 252.1 for sorafenib, and m/z 268.23 to 116.03 for metoprolol, which served as an internal standard. Plasma samples were deproteinized with 4 volumes of acetonitrile (Nacalai Tesque) and the supernatants were used for analysis.

The Microsoft Excel software was used to analyze the obtained data. Means, standard deviations (SD), and relative standard deviations (RSD) were calculated. For the analysis of MS data, the Waters QuanLynx software was used. Each described sample was prepared in triplicate and analyzed at least three times.

UPLC–MS/MS was used for the analysis and determination of DON and ZEN. The methods for extraction, clean-up and determination of DON and ZEN from the cultures was based on previously published work [9 (link)] with slight modifications. Briefly, 5 mL culture filtrate was extracted with 20 mL of acetonitrile for 30 min using an automatic shaker. After centrifugation, 8 mL of the supernatant was passed through a MycoSep 226 Aflazon + multifunctional column (Romer Labs, Inc. Union, MO, USA) and 4 mL of the purified extract was evaporated to dryness under a stream of nitrogen. The residue was then dissolved in 1 mL of methanol–water (50/50, v/v), followed by filtering, and was subsequently used for analysis. Data acquisition and processing were performed using MassLynx v4.1 and Quanlynx (Waters, Milford, MA, USA).

Dissected brain tissue was snap frozen on dry ice and stored at −80°C until analysis. Brain regions were prepared according to the protocol for IDO activity in brain tissue. Samples were prepared by mixing 450 μg brain protein 1:2 (v/v) with methanol containing 2% acetic acid and then centrifuging the mixture at 12,000×g for 10 minutes at 4°C. Supernatants were filtered through a Phenomenex Phree Phospholipid extraction column and a 0.2 μm filter. The samples were then run on a Waters Acquity UPLC-MS/MS with a methanol/2% acetic acid mobile phase gradient on a 2 × 100−mm Waters BEH C¹⁸ column. The sample (5 μl) was injected with a total flow rate of 0.3 ml/min and a total run time of 4.5 minutes. Analytes were verified using two M+H parent-daughter transitions. Tryptophan parent m/z = 205.13 and daughter m/z = 118.03 and 146.07; kynurenic acid parent m/z = 190.07 and daughter m/z = 88.96 and 116.03; kynurenine parent m/z = 209.04 and daughter m/z = 94.02 and 146.03; 3-HK parent m/z = 224.98 and daughter m/z = 110.04 and 162.05. Concentrations were quantified using QuanLynx (Waters) software from serial dilutions of standards.