About the Platform
The NIEHS untargeted metabolomics platform is discovery-oriented, measuring chemicals (<1000 Da) of endogenous, microbial, and xenobiotic origin in a variety of matrices including cell lysates, tissue homogenates, and biofluids (e.g., plasma, urine).
- Broad coverage of the metabolome.
- Chemicals/metabolites are reported as features based on their measured values (m/z, retention time, etc.) (see Frequently Asked Questions).
- High annotation confidence for ~500 metabolites that we have analyzed in-house using authentic chemical standards (53KB).
- Numeric values (relative abundance) reported for all features in a sample.
- These features are annotated (assigned a tentative chemical identity) using MS/MS spectral database matching. Typical annotation rate is 5 – 10% (often amounting to several hundred chemicals), with an estimated 5 – 10% false discovery rate.
Analytical Approach
Ultra-high performance liquid chromatography – high-resolution tandem mass spectrometry (UHPLC – HRMS/MS). Ultra-high performance liquid chromatography (UHPLC) is used to separate chemicals, prior to mass spectrometric analysis, based on physical and chemical characteristics such as polarity. High-resolution mass spectrometry (HRMS) is used to compute molecular formulae and isotope patterns. Tandem mass spectrometry (MS/MS), specifically product ion scans, yields structural information. Data are collected in the positive and negative ionization mode.
Accepted Samples
The types of samples accepted for analysis are listed below, along with the recommended (and minimum) material required. Additional sample types are welcome but will be accepted after consultation, on a case-by-case basis. Further, if minimum material amounts cannot be met, please contact us for guidance. For most sample types, we recommend a minimum of ~4 – 5 biological replicates; however, replicates and additional important technical issues relating to sample preparation can be discussed during initial project consultation.
| Sample Types | Minimum Material | Recommended Material |
|---|---|---|
| Biofluids | ||
| Serum or Plasma | 50 microliters | 200 microliters |
| Urine | 50 microliters | 200 microliters |
| Saliva | 50 microliters | 200 microliters |
| Tissues | ||
| Kidney, liver, ovary, ears, brain, heart, retina, etc. | 5 mg tissue | 20 – 25 mg tissue |
| Tumors | 5 mg tissue | 20 – 25 mg tissue |
| Drosophila (whole organism) | 5 organisms | 10 organisms |
| Tissues | ||
| Mammalian cells (cell lysates) | 1 – 2 million cells | >2 million cells |
| Yeast cells (cell lysates) | 1 – 2 million cells | >2 million cells |
| Miscellaneous | ||
| Botanical extracts | 50 microliters | 200 microliters |
| Plant material | 5 mg tissue | 20 – 25 mg tissue |
What is Included?
- All projects include consultation on experimental design. Pilot samples included at no cost at the discretion of the Core.
- Analytical measurement of samples by UHPLC-HRMS/MS.
- Processing of raw data via formatting, quality control, and data cleaning.
- Sample preparation handled by the sample submitter (protocols will be provided) or by Core personnel on a case-by-case basis.
- Specific deliverables can be found in the Project Tier section.
Project Tiers
We offer two tiers of projects with different deliverables and rates; please see below for details.
Tier 2 – Collaborative
Most popular and highly recommended for all metabolomics projects.
Deliverables
- de novo annotation of the top-n most significant unannotated features.
- Initial statistical analysis based on primary hypothesis to guide interpretation, including calculation of fold-changes, univariate statistics, and multivariate statistics (.txt) and corresponding plots (.pdf and/or .png).
- Processed data (.txt) and corresponding list of annotations (.txt).
- Raw data (vendor files).
Rates
| Number of Samples | Summarized Cost |
|---|---|
| 1 – 100 | $100 / sample |
| 101 – 300 | $10,000+ $75 / sample over 100 samples |
| 301+ | Contact Core for Approval |
Tier 1 – Service
Recommended for experts in metabolomics data analysis.
Deliverables
- Processed data (.txt) and corresponding list of annotations (.txt).
- Raw data (vendor files).
Rates
| Number of Samples | Summarized Cost |
|---|---|
| 1 – 100 | $75 / sample |
| 101 – 300 | $7,500+ $60 / sample over 100 samples |
| 301+ | Contact Core for Approval |
Conditions
- Samples must be prepared according to Metabolomics Core specifications. Core reserves the right to refuse samples which do not adhere to guidance.
- We kindly request that you consider co-authorship for Core personnel (for Tier 2 – Collaborative projects) or acknowledgement (for Tier 1 – Service projects) in any resulting work (manuscript, presentation, etc.).
Frequently Asked Questions
What is a feature?
A feature is a unique set of measured values (m/z, retention time, MS/MS) that is putatively thought of as a unique chemical. Algorithms detect and report features in an unsupervised fashion, therefore, there is an imperfect correlation between features and chemicals.
What is an annotation?
Features are annotated, i.e., provided a chemical name, via spectral matching between acquired MS/MS spectra from samples and MS/MS spectra in public and commercial libraries. The annotation is not certain and should be thought of as a putative name that requires manual confirmation of evidence supporting the annotation. The certainty of an annotation can be promoted to an identification using authentic chemical standards analyzed in an identical manner to samples.
What chemicals will be annotated in my sample?
There are multiple factors that determine if a chemical will be annotated. The chemicals that are most likely to be identified are those which occur in well characterized materials (e.g., blood or urine), chemicals which are abundant (amount or concentration), chemicals which are stable, and chemicals for which authentic chemical standards can be purchased or synthesized.
How will annotations be reported?
The annotations will be reported according to the Metabolomics Standards Initiative (MSI) levels, which rank 1 – 4 (1 is the most confident annotation). These levels consider numerous physical and spectral characteristics of a chemical (summarized below). Our standard approach produces MSI level 2 annotations. We can, in limited cases, promote MSI level 2 annotations to MSI level 1 (aka identification); however, the unambiguous identification of stereoisomers and/or constitutional isomers may not be possible.
How many annotations should I expect?
The number of annotations will vary tremendously based on the material being analyzed; however, one should reasonably expect 5 – 10% of all MS features having a putative annotation (level 2 or greater) for well characterized materials (e.g., blood or urine). Uncommon or extremely complex starting materials will have lower annotation rates.
I know this chemical is present in my sample. Why was it not annotated?
While untargeted metabolomics aims to detect all chemicals present in a sample, there are multiple steps that can introduce chemical selectivity including the sample processing, chromatography, and ionization. It might be that the amount is insufficient to detect – even with the sensitivity of a mass spectrometer – or if the chemical is unstable (oxidation, pH, thermal, etc.), it may not be observed. A universal answer is hard to provide; please contact us and we can assist.
What to do when an MS feature does not have an annotation?
Unfortunately, this occurs often given the rather limited ability to confidently annotate all chemicals within a sample. However, there are several remedies to address this issue (de novo annotation via manual interpretation and in silico prediction tools), but prioritization is critical as a single annotation can take anywhere from minutes to months of work. We approach de novo annotation work by prioritizing what is statistically significant or important to your work, rather than focusing on giving every MS feature an annotation regardless of importance.
Contact
All initial project consultations should be directed to the Trans-NIH Metabolomics Core Coordinator, Kirsten E. Overdahl, Ph.D.
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Alan K. Jarmusch, Ph.D.
Director, Metabolomics Core Facility (MCF) -
Tel 984-287-4523
alan.jarmusch@nih.gov
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Kirsten E. Overdahl, Ph.D.
Chemist -
Tel 984-287-4562
kirsten.overdahl@nih.gov
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