Summary of Study ST001361
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000931. The data can be accessed directly via it's Project DOI: 10.21228/M8BT4S This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001361 |
| Study Title | Serum tryptophan metabolomics in CKD |
| Study Type | MS quantitative analysis |
| Study Summary | Serum was processed using a targeted metabolomics platform for quantifying tryptophan metabolites as a number of these metabolites are well establish uremic toxins. |
| Institute | University of Florida |
| Last Name | Ryan |
| First Name | Terence |
| Address | 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA |
| ryant@ufl.edu | |
| Phone | 352-294-1700 |
| Submit Date | 2020-04-02 |
| Num Groups | 2 |
| Total Subjects | 16 |
| Num Males | 8 |
| Num Females | 8 |
| Raw Data Available | Yes |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-12-31 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Combined analysis:
| Analysis ID | AN002265 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex |
| Column | ACE 5 C18-300 (100 x 2.1mm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | ng/ml |
Chromatography:
| Chromatography ID: | CH001664 |
| Chromatography Summary: | Twenty-five microliters of each mouse serum was spiked with 5 µL internal standards (IS) solution consisted of tryptophan ¹³C₁₁, serotonin D4, kynurenine D4, kynurenic Acid D5, xanthurenic acid D4, anthranilic acid ¹³C₆, indoxyl sulfate ¹³C₆, p-cresol sulfate D7 and 3-indole-acetate D7. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization for all with a mass resolution of 35,000 at m/z 200 as separate injections. Tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid and anthranilic acid were quantified in the positive ionization while indoxyl sulfate, p-cresol sulfate and 3-indole-acetate were analyzed in negative ionization. Separation was achieved on an ACE 18-PFP 100 x 2.1 mm, 2 µm column using a gradient with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. The flow rate was 350 µL/min with a column temperature of 25°C and injection volume of 2 µL. Run time was 20.5 min. A 9-point calibration curve and QC samples were prepared for targeted quantitation of tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate, p-cresol sulfate and 3-indole-acetate. 20 µL of each calibrator and QCs were supplemented with 5 µl indoxyl sulfate. Peak areas of each analyte and corresponding internal standard in the calibrator, QCs and samples were integrated using Xcalibur 4.0. A calibration curve was generated by plotting nominal concentration of the analyte in the calibrators versus peak area ratio of analyte and IS. QCs and samples were quantitated against the calibration curve. |
| Instrument Name: | Thermo Dionex |
| Column Name: | ACE 5 C18-300 (100 x 2.1mm) |
| Column Temperature: | 25C |
| Flow Rate: | 350ul/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
