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MB Sample ID: SA005133
| Local Sample ID: | ConstantVolume_UT_1_1 |
| Subject ID: | SU000111 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and Female |
| Human Race: | Hispanic, Aisan, Caucasian |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000111 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and Female |
| Human Race: | Hispanic, Aisan, Caucasian |
| Species Group: | Human |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| ConstantVolume_UT_1_1 | SA005133 | FL001329 | Urease pre-treatment | Urease Treatment |
| ConstantVolume_UT_1_1 | SA005133 | FL001329 | 100 | Volume Urine (µL) |
| ConstantVolume_UT_1_1 | SA005133 | FL001329 | N/A | gender |
| ConstantVolume_UT_1_1 | SA005133 | FL001329 | Urease | Solution Added |
| ConstantVolume_UT_1_1 | SA005133 | FL001329 | N/A | ethnicity |
Collection:
| Collection ID: | CO000094 |
| Collection Summary: | Approval for the conduct of this programmatic research was obtained from the Pacific Northwest National Laboratory Institutional Review Board. Urine samples from consenting male and female donors (n = 20 each, Supplemental Table S1) after an overnight fast were purchased from Bioreclamation, LLC (Hicksville, NY) and received frozen on dry ice and deidentified. To create a uniform sample for Experiments 1 and 2 (see below), aliquots from each individual sample were pooled, realiquoted, and stored at -80C until used. |
| Sample Type: | Urine |
| Storage Conditions: | -80° C |
Treatment:
| Treatment ID: | TR000112 |
| Treatment Summary: | Varying Volume with Urease / Male vs. Female with Urease / Constant Volume with Urease / Constant Volume with 100 µL Water |
| Treatment Protocol Comments: | To evaluate whether the effects of urease pre-treatment of urine varied with the volume of urine prepared, we compared the urine metabolite profiles from pooled urine after pre-treatment with urease (UT) and after no treatment (NT) using various volumes of urine. For this, several volumes (5, 10, 25, 50, and 100 µL) of the pooled urine sample were incubated (n = 3, each) with 100 µL of a 1 mg/mL solution of urease or were not subjected to any treatment. / Finally, to evaluate whether any artifacts introduced by urease pre-treatment on the urinary metabolome interfered with the ability to distinguish between comparative samples, we compared the metabolite profiles from individual male and female urine samples after pre-treatment with urease or after no treatment (previous metabolomics studies of male and female urines (Pasikanti et al., 2008; Slupsky et al., 2007; Saude et al., 2007; Psihogios et al., 2008) have reported differences in metabolite levels). For this, 50 µL aliquots of individual male and female urine samples (n = 20, each) were blocked, randomized, and then incubated with 50 µL of a 1 mg/mL solution of urease (UT) or were not subjected to any treatment (NT), each as described above. / To initially evaluate the effects of urease pre-treatment on the urinary metabolome, we compared the urine metabolite profiles from pooled urine after pre-treatment with urease, water, or no treatment at all. For this, 100 uL aliquots of the pooled urine sample were incubated (n = 5, each) with 100 uL of a 1 mg/mL solution of urease (Sigma-Aldrich catalog number U4002) prepared in water (urease-treated; UT) or an equal volume of water alone (water-treated; WT) for 30 min at 37 C with mild shaking (500 rpm). Identical aliquots (n = 5) were not subjected to any treatment (no treatment; NT) and allowed to sit at room temperature for 30 min. / To initially evaluate the effects of urease pre-treatment on the urinary metabolome, we compared the urine metabolite profiles from pooled urine after pre-treatment with urease, water, or no treatment at all. For this, 100 uL aliquots of the pooled urine sample were incubated (n = 5, each) with 100 uL of a 1 mg/mL solution of urease (Sigma-Aldrich catalog number U4002) prepared in water (urease-treated; UT) or an equal volume of water alone (water-treated; WT) for 30 min at 37 C with mild shaking (500 rpm). Identical aliquots (n = 5) were not subjected to any treatment (no treatment; NT) and allowed to sit at room temperature for 30 min. |
| Treatment: | Abiotic |
| Treatment Compound: | Urease / Urease / Urease / Water |
| Treatment Dose: | 1 mg/ml / 1 mg/ml / 1 mg/ml / -- |
| Treatment Dosevolume: | 100 µL / 50 µL / 100 µL / 100 µL |
| Human Fasting: | Overnight Fast |
Sample Preparation:
| Sampleprep ID: | SP000107 |
| Sampleprep Summary: | concomitant protein precipitation with cold methanol, vortexing, centrifugation, supernatent dried in vacuo, stored at -80° c, chemical derivitization |
| Sampleprep Protocol Comments: | Metabolites were extracted with concomitant protein precipitation by addition of 1 mL of cold (-20 C) methanol with vortexing for 30 s, and precipitated proteins were removed by centrifugation at 15,000xg for 10 min at 4 C. The supernatants were transferred to glass autosampler vials and then dried in vacuo prior to chemical derivatization. If the extracts could not be immediately derivatized an d analyzed by GCMS, then they were stored at -80C. Dried metabolite extracts were chemically derivatized using a modified version of the protocol used to create FiehnLib (Kind et al., 2009). Briefly, dried metabolite extracts were dried again to remove any residual water if they had been stored at -80°C. To protect carbonyl groups and reduce the number of tautomeric isomers, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by vortexing for 30 s and incubation at 37°C with generous shaking (1000 rpm) for 90 min. At this point, the sample vials were inverted one time to capture any condensation of solvent at the cap surface, followed by a brief centrifugation at 1000×g for 1 min. To derivatize hydroxyl and amine groups to trimethylsilyated (TMS) forms, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were then added to each vial, followed by vortexing for 10 s and incubation at 37°C with shaking (1000 rpm) for 30 min. Again, the sample vials were inverted one time, followed by centrifugation at 1000×g for 5 min. The samples were allowed to cool to room temperature and were analyzed in the same day. |
| Processing Method: | precipitation, centrifugation |
| Processing Storage Conditions: | dried in vacuo, stored at -80° C |
| Extraction Method: | concomitant protein precipitation by addition of 1 mL of cold (-20 C) methanol with vortexing and centrifugation |
| Extract Enrichment: | dried in vacuo |
| Extract Storage: | -80° C |
| Sample Resuspension: | 20 µL of methoxyamine in pyridine (30 mg/mL) |
| Sample Derivatization: | 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) |
Combined analysis:
| Analysis ID | AN000146 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890A |
| Column | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5975C |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH000104 |
| Chromatography Summary: | Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation |
| Chromatography Comments: | Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min. |
| Instrument Name: | Agilent 7890A |
| Column Name: | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| Flow Rate: | 0.450.5 mL/min |
| Injection Temperature: | 250 C |
| Sample Injection: | 1 L, splitless |
| Analytical Time: | 37.5 min |
| Oven Temperature: | 60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C |
| Sample Syringe Size: | 10 L |
| Chromatography Type: | GC |
MS:
| MS ID: | MS000122 |
| Analysis ID: | AN000146 |
| Instrument Name: | Agilent 5975C |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis. |
| Ion Mode: | POSITIVE |
| Scan Range Moverz: | 50-550 m/z |
