Clinical Participants
From March 2019 to December 2019, TS patients and healthy controls were recruited from Nanjing Hospital of Chinese Medicine, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine. TS patients were diagnosed by the Diagnostic and statistical manual of mental disorders (DSM-5®)[15]. Healthy controls without TS and other known infections were included. All participants provided written informed consent. Finally, the total 49 participants have been recruited: 30 TS samples and 10 healthy controls for the untargeted UHPLC-Q-TOF/MS metabolomics analysis, the average age of the TS patients was 7.9 years, and 21 males (70.0%) and 9 females (30.0%) were included, the mean age of the healthy controls was 9.2 years, and 6 males (60.0%) and 4 females (40%) were included (sex p=0.559, age p=0.211); Another 35 TS samples and 14 healthy controls(include 30 TS samples and 10 healthy controls in untargeted analysis) for targeted UHPLC/MS/MS analysis, the average age of the TS patients was 8.086 years, and 24 males (69.57%) and 11 females (31.43%) were included, the mean age of the healthy controls was 9.5 years, and 9 males (64.29%) and 5 females (35.71%) were included(sex p=0.773, age p=0.064). There was no statistical significance in both age and gender in TS and healthy controls.
Chemicals and reagents
HPLC grade methanol (Thermo, A456-4); HPLC grade acetonitrile (Merck, 1499230-935); ammonium acetate (Sigma, 70221); ammonium hydroxide (Fluka). Analytical standards including L-Glutamic acid hydrochloride, D-Ornithine monohydrochloride, L-Ornithine monohydrochloride, D-Homoproline, D-Proline, L-Arginine, L-Pipecolic acid, L(-)- Carnitine were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Millipore-Q Water Purification System (Millipore, Germany) provided ultra-high purity water. All other chemicals and reagents were obtained from Merck Sigma-Aldrich (KGaA, Darmstadt, Germany).
Sample preparation
Blood samples were collected from each participant, then the samples were centrifuged 15 min(3000×rcf, 4°C) within 1 hour of collection. Each aliquot (1mL) of the plasma samples were stored at -80°C until the UHPLC-Q-TOF/MS and UHPLC/MS/MS processing. For the untargeted analysis, the plasma samples were thawed at 4°C. 400 μL of methanol/acetonitrile (1:1, v/v) were added to 100μL of plasma After 60 sec. of the vortex, the mixture was stored at -20°C for 1 hour to remove protein, and then centrifuged at 14,000×rcf for 20 min at 4°C. The supernatants were subjected to UHPLC-Q-TOF/MS. -Quality control(QC) samples: 10μL of each plasma samples were mixed and treated in the same way as plasma samples. The QC samples were inserted in every 8 samples to monitor the system stability of UHPLC-Q-TOF/MS. For the targeted analysis, each plasma sample was thawed at 4°C for 30 min and 200μL aliquots were mixed with 200μL of methanol. Then the tubes were vortexed for 60 sec and then centrifuged for 15 min(13,000×rcf, 4°C). Preparation of standard solution: each 1 mg analytical standard was dissolved in methanol (1mg/ml) and then diluted in methanol 10μg/ml. The supernatant was transferred to an autosampler vial and a UHPLC/MS/MS method was used for the detection.
UHPLC-Q-TOF/MS processing and data analysis
UHPLC-Q-TOF/MS analysis was performed on an Agilent 1290 Infinity LC system (Agilent Technologies, Santa-Clara, California, USA), equipped with an AB SCIEX Triple TOF 5600 System (AB SCIEX, Framingham, MA, USA) in both positive and negative modes. Chromatographic separation was performed on ACQUITY HSS T3 1.8 µm (2.1 × 100 mm) columns. The column temperature was kept at 25°C and the flow rate was 0.3 mL/min. The UHPLC system consisting of water with 25mM ammonium acetate with 25 mM ammonia (solvent A) and acetonitrile (solvent B). The gradient profile used was optimized as follows: 0-1min,95%B; 1-14min, linearly changed to 65%B; 14-16min, linearly changed to 40%B; 16-18min,40%B; 18-18.1min, linearly increased to 95%B; 18.1-23min, 95%B. The sample injection volume was 2μL.
Electrospray ionization (ESI) source conditions were set as follows: ion source gas 1(gas 1) was 60 psi; ion source gas 2(gas 2) was 60 psi; curtain gas(CUR) was 30 psi; source temperature was set to 600 °C; ion spray voltage floating(ISVF) was 5000V (+) and −5000V (−). Information-dependent acquisition (IDA) is a product ion scan mode based on artificial intelligence, which was used to detect and identify MS/MS spectra. The parameters were set as follows: declustering potential (DP) was set to 60V (+), and −60V (−); collision energy was 35±15 eV; exclude isotopes within 4 Da, candidate ions to monitor per cycle: 6.
The raw data was generated by the Proteo Wizard MS converter tool, and processed by XCMS online software (https://xcmsonline.scripps.edu/landing_page.php?pgcontent=mainPage), including the nonlinear alignment, automatic integration, and peak extraction. After being normalized and integrated, MetaboAnalystR (3.0.3)[16] was employed for the statistical analysis (including PLS-DA analysis, hierarchical cluster analysis, Fold Change Analysis, and T-tests) and bioinformatics (pathway enrichment analysis) (www.metaboanalyst.ca). Significance was analyzed using Adjusted p-value < 0.05 and |logFC| >1. For pathway enrichment analysis, two enrichment algorithms integrated mummichog and GSEA were used, and P < 0.05 was considered statistically significant.
Selection of metabolites for targeted metabolomics
For the selection of biomarkers, the affected metabolic pathway containing abundant affected metabolites was the principal criterion. The preliminary identification of these metabolites was conducted by matching with the self-constructed databases (the secondary spectral library of standard samples established in the same experimental system, about 2500 kinds). In addition, The similarity values for the accuracy of compound identification and the number of differentially metabolites detected in each test sample were also important to reference factors[17]. Then the selected metabolites were identified by standards and tandem mass spectrometry.
Sample processing and targeted UHPLC/MS/MS analyses
The targeted validation of metabolites was carried out on an Agilent 1290 Infinity LC system (Agilent Technologies, Santa-Clara, California, USA) equipped with the Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies). The electrospray ionization(ESI) source was set in positive ion modes. Chromatographic separation was implemented on Agilent ZORBAX Hilic Plus (Agilent, USA) (50 × 2.1mm, 1.8μm) at a column temperature of 30oC, a flow rate of 0.3 mL/min. The mobile phase A consisted of solvent A (methanol), and solvent B (0.1% formic acid in water). The gradient profile was performed as follows: 0-1min,5%B; 1-2min, linearly increased to 90%B; 2-3min,90%B; 3-3.1 min, returned to 5%; 3.1-4min,5%B. The sample injection volume was 5μL. The optimized MS settings were: capillary voltage, 4000v(+), 3500v(-); the dry gas flow, 10 L·min~(-1), and the drying gas temperature, 350oC. The quantitation was performed using multiple reaction monitoring (MRM) mode. The concentrations data of individual metabolites were calculated directly by UHPLC-MS/MS analysis (nmol/g).
In the UHPLC/MS/MS targeted analyses, the Student's t-test and the Mann-Whitney U test was used for comparisons between healthy and TS patients. All statistical analyses were analyzed by GraphPad Prism8 (GraphPad Software corporation, California, USA). Statistically, significance was defined by P<0.05 (two-tailed). And the area under the ROC curves (AUC) was calculated to further analyze the differential metabolites.