Return to study ST002984 main page
MB Sample ID: SA323838
Local Sample ID: | 56 |
Subject ID: | SU003097 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Combined analysis:
Analysis ID | AN004903 | AN004904 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Ion exchange | Reversed phase |
Chromatography system | Thermo Dionex ICS-5000+ | Shimadzu Nexera X2 |
Column | Dionex IonPac AS11-HC (250 x 2mm, 4um) | Sigma-Aldrich Discovery HS F5-3 (150 x 2.1 mm, 3 um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Triple quadrupole |
MS instrument name | Thermo Q Exactive Focus | LCMS-8060, Shimadzu Corporation |
Ion Mode | UNSPECIFIED | UNSPECIFIED |
Units | Peak area/Internal Standard | Peak area/Internal Standard |
Chromatography:
Chromatography ID: | CH003698 |
Chromatography Summary: | Metabolites were detected using an orbitrap-type MS instrument (Q-Exactive focus; Thermo Fisher Scientific) connected to a high-performance IC system (ICS-5000+, Thermo Fisher Scientific) that enabled highly selective and sensitive metabolite quantification owing to the IC separation and Fourier transfer MS principle. The IC instrument was equipped with an anion electrolytic suppressor (Dionex AERS 500; Thermo Fisher Scientific) to convert the potassium hydroxide gradient into pure water before the sample entered the MS instrument. Separation was performed using a Dionex IonPac AS11-HC 4 μm particle size column (Thermo Fisher Scientific). The IC flow rate was 0.25 mL/min, supplemented post-column with 0.18 mL/min makeup flow of MeOH. The potassium hydroxide gradient conditions for IC separation were as follows: from 1 mM to 100 mM (0–40 min) to 100 mM (40–50 min) and to 1 mM (50.1–60 min) at a column temperature of 30 °C. The mass spectrometer was operated in the ESI-positive and negative mode with polarity switching, for all detections. A full mass scan (m/z 70–900) was performed at a resolution of 70,000. The automatic gain control target was set at 3 × 106 ions, and the maximum ion injection time was 100 ms. The source ionization parameters were optimized with a spray voltage of 3 kV, and other parameters were as follows: transfer temperature, 320 °C; S-Lens level, 50; heater temperature, 300 °C; sheath gas, 36; and aux gas, 10. |
Instrument Name: | Thermo Dionex ICS-5000+ |
Column Name: | Dionex IonPac AS11-HC (250 x 2mm, 4um) |
Column Temperature: | 30 °C |
Flow Gradient: | From 1 mM to 100 mM (0–40 min) to 100 mM (40–50 min) and to 1 mM (50.1–60 min) |
Flow Rate: | 0.25 mL/min, supplemented post-column with 0.18 mL/min makeup flow of MeOH |
Solvent A: | The potassium hydroxide gradient conditions |
Solvent B: | N/A |
Chromatography Type: | Ion exchange |
Chromatography ID: | CH003699 |
Chromatography Summary: | Cationic metabolite concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In essence, we employed a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (LCMS-8060, Shimadzu Corporation, Kyoto, Japan) operated in both positive and negative-ESI and in multiple reaction monitoring (MRM) modes. Analyte separation was achieved on a Discovery HS F5-3 column (2.1 mm I.D. × 150 mm L, 3 μm particle size; Sigma-Aldrich, St. Louis, MO, USA) through a gradient elution with mobile phase A (0.1% formate) and mobile phase B (0.1% acetonitrile). The elution profile was as follows: 100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min), and 100:0 (20–25 min), with a constant flow rate of 0.25 mL/min and a column oven set at 40 °C. |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Sigma-Aldrich Discovery HS F5-3 (150 x 2.1 mm, 3 um) |
Column Temperature: | 40 °C |
Flow Gradient: | 100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min), and 100:0 (20–25 min) |
Flow Rate: | 0.25 mL/min |
Solvent A: | 0.1% formate |
Solvent B: | acetonitrile with 0.1% formate |
Chromatography Type: | Reversed phase |