Summary of Study ST000092
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 PR000084. The data can be accessed directly via it's Project DOI: 10.21228/M8PK5V This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000092 |
| Study Title | A statistical analysis of the effects of urease pre-treatment on the measurement of the urinary metabolome by gas chromatographymass spectrometry |
| Study Type | Analytical Comparison |
| Study Summary | Urease pre-treatment of urine has been utilized since the early 1960s to remove high levels of urea from samples prior to further processing and analysis by gas chromatographymass spectrometry (GCMS). Aside from the obvious depletion or elimination of urea, the effect, if any, of urease pre-treatment on the urinary metabolome has not been studied in detail. Here, we report the results of three separate but related experiments that were designed to assess possible indirect effects of urease pre-treatment on the urinary metabolome as measured by GCMS. In total, 235 GCMS analyses were performed and over 106 identified and 200 unidentified metabolites were quantified across the three experiments. The results showed that data from urease pre-treated samples (1) had the same or lower coefficients of variance among reproducibly detected metabolites, (2) more accurately reflected quantitative differences and the expected ratios among different urine volumes, and (3) increased the number of metabolite identifications. Overall, we observed no negative consequences of urease pre-treatment. In contrast, urease pre-treatment enhanced the ability to distinguish between volume-based and biological sample types compared to no treatment. Taken together, these results show that urease pre-treatment of urine offers multiple beneficial effects that outweigh any artifacts that may be introduced to the data in urinary metabolomics analyses. |
| Institute | Pacific Northwest National Laboratory |
| Department | Biological Separation and Mass Spectrometry |
| Last Name | Metz |
| First Name | Thomas |
| thomas.metz@pnnl.gov | |
| Submit Date | 2014-06-25 |
| Num Groups | 6 |
| Total Subjects | 235 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | cdf |
| Uploaded File Size | 3.0 G |
| Analysis Type Detail | GC-MS |
| Release Date | 2014-08-07 |
| Release Version | 1 |
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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) |
