The Five Key Elements of a Successful Metabolomics Study

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1 The Five Key Elements of a Successful Metabolomics Study

2 Metabolomics: Completing the Biological Picture Metabolomics is offering new insights into systems biology, empowering biomarker discovery, and advancing research across the life sciences. Metabolomics is the systematic measurement of the low molecular weight biochemicals (~ Da) known as metabolites. Nearly every variable, from genes and microbiota to disease and diet, exerts influence by subtly altering metabolite levels. In this way, metabolites are sensitive indicators of homeostatic imbalances. Measuring metabolites using metabolomics improves our understanding of how disease, drug exposure, genetics, the microbiome, diet, and lifestyle all influence the phenotype. 2 The 5 Key Elements of a Successful Metabolomics Study 1

3 Leveraging Metabolomics to Advance Research This brief e-book will provide you with the Five Key Elements of a successful metabolomics study: 1. Define a Clear Objective 2. Use Strong Study Design Elements Researchers across multiple areas of the life sciences are using metabolomics to answer key research questions and advance programs in translational science, biomarker identification, and the study of disease and treatment mechanisms. While metabolomics has proven itself to be an incredibly valuable research tool, metabolomics studies also have unique challenges to consider. 3. Power Your Study for Success 4. Choose The Right Profiling Technology for Your Goals 5. Have a Data Interpretation Plan in Place The 5 Key Elements of a Successful Metabolomics Study 2

4 1. Define a Clear Objective The first and most critical step in any successful scientific study is to clearly define the study s objective. Are you seeking general information to help you form a hypothesis? Do you have a hypothesis in mind that you wish to validate? Are you hoping to discover biomarkers for a disease? Do you want to understand the MOA of a potential drug candidate? The Metabolon Advantage: Metabolon offers every investigator study design assistance from an experienced Ph.D. scientist. The 5 Key Elements of a Successful Metabolomics Study 3

5 Define Your Approach A clear objective is critical to identifying the ideal metabolomics approach for your study. For example, if you want to understand a variable s impact on the subject s entire metabolic system, you probably want to employ a discovery metabolomics approach, which surveys all of the metabolites from across the whole metabolome. On the other hand, if you already have some understanding of the metabolites implicated in your study, you may want to narrow your focus and home in on specific metabolite classes (such as lipids), specific metabolic pathways (such as the glycolysis pathway), or a specific metabolite biomarker. Discovery Metabolomics Whole-metabolome Profile Disease Characterization Target Discovery/Validation Biomarker Discovery Translational Studies Focused Profiling Pathway Regulation Markers of Efficacy Monitor Individual Response Translational Studies Biomarker Assays Trial Enrollment Clinical Diagnostics The 5 Key Elements of a Successful Metabolomics Study 4

6 2. Use Strong Study Design Elements Strong study design elements are central to uncovering biologically significant results. Select the appropriate sample matrix (or combination of matrices) Collect adequate exposures (dose and time of collection) Employ controls for each tested variable don t skimp! Take steps to minimize excess variation maintain consistency! A Tip from Metabolon: The more inherent variation you can control for, the fewer samples that are required for the study. The 5 Key Elements of a Successful Metabolomics Study 5

7 Sample Matrix & Collection SelectYour Sample Matrix Depending on what you are hoping to uncover, select an appropriate sample matrix or matrices. Try to use the matrix or combination of matrices that are closest to the system you are attempting to study. For example, when studying the effect of drug exposure on the brain, a CSF sample will provide significantly more information than a urine sample. On the other hand, urine would be an appropriate matrix if you are attempting to study a treatment effect on kidney function. When using animal models it may be advisable to also include organ samples, which could improve understanding of the physiology s precise site of action. Collect Adequate Exposures Sampling at only a single exposure point is a gamble and may result in too few metabolic changes to derive any biomarkers or enhanced understanding. At the other extreme, a single point sample may reveal so many changes that it can be difficult to assign cause and effect. Multi-exposure point studies, such as a time-course study or a multi-dose study, increases your chance of seeing meaningful variations. A good rule of thumb is to collect samples at mild, moderate and severe exposures to ensure that all salient cause and effect changes are captured. The 5 Key Elements of a Successful Metabolomics Study 6

8 Variables & Controls A Control for Every Variable Sometimes our desire for robust results around a research project makes us overly ambitious. It s important to avoid the temptation of testing multiple variables unless you are prepared to have each variable accompanied by rigorous controls. Embedding too many questions in a study without sufficient controls built around each questions can leave you with a lot of data and few meaningful answers. Only incorporate as many questions and variables as you can realistically test with adequate controls. Minimize Excess Variation For every study, you need to consider how much variance is inherent in the cohort or study samples. For example, inbred rodent model have significantly less variability than human study subjects with different ages, genders, genotypes, BMIs and lifestyles. To accommodate the off-target variables, collect adequate samples, employ strong controls, and attempt to balance the groups where appropriate. Also, consistency in sample collection and handling is essential. Having rigorous collection and handling protocols minimizes variance so that you can obtain meaningful results with fewer samples. Remember: The more inherent variation you can control for, the fewer samples that are required for the study. The 5 Key Elements of a Successful Metabolomics Study 7

9 3. Power Your Study For Success Strong study design can deliver biologically significant results, but a well-powered study can provide statistically significant results. Study Subjects Per Group Cell Culture Small Animals Human Studies Optimal >7 >10 >50 Rigorous Acceptable Fewer Required Strong phenotype or treatment effect (toxicology study) Repeated sampling from the same subject Multiple time points Multiple doses of a drug/inhibitor More Required Subtle phenotype or treatment effect (dietary supplements, exercise-induced changes Mixed populations of subjects (mixed gender, wide-ranging age or BMI) Multiple-site collections The 5 Key Elements of a Successful Metabolomics Study 8

10 Suggested Sample Type & Amount for Adequate Power Sample Quantities Recommended for Optimal Results: Biological Fluids: 100 μl Cells: 100 μl pellet Cell-Based Studies t1 t2 Vehicle Control 5 5 Drug Dose Drug Dose Small Animal Studies Chow HFD WT 8 8 KO 8 8 Overexpression 8 8 t Tissues: 100 mg Human Studies Control Case The 5 Key Elements of a Successful Metabolomics Study 9

11 4. Choose the Right Technology for Your Goals Metabolomics requires a different and more sophisticated profiling platform than many other types of omic research (such as genomic or proteomic research). Metabolites, which are small and highly abundant, are also extremely chemically diverse, and they vary substantially in their molecular weight and solubility. This makes it difficult for a single analytical method to accurately measure all of the metabolites present in a sample. Profiling Platform In order to have meaningful study results, you should select a metabolomics platform (screening technology) with both advanced instrumentation and sophisticated informatics. Sensitive and precise instrumentation is necessary to detect all of the low-level metabolites present in a sample, while sophisticated informatics help rapidly and accurately identify and quantify these metabolites. The 5 Key Elements of a Successful Metabolomics Study 10

12 The Right Instrumentation There are many approaches to generating metabolomics data. Some of the most popular approaches include liquid or gas chromatography coupled to mass spectrometry (LC/MS, GC/MS), nuclear magnetic resonance (NMR) spectroscopy, and capillary electrophoresis (CE) chromatography mass spectrometry methods. Each approach has pros and cons, and some may be more suited to your study than others. NMR, for example, does not destroy the sample, but it has very limited breadth of metabolite coverage. CE allows you to profile with smaller sample quantities and great resolving power, but it has limitations for detecting neutral metabolites. UPLC/MS technology is among the most sensitive and highly-resolving metabolomics technologies. LC-MS offers a broad and sensitive screening of most metabolite classes, while GC-MS and LC-MS+ HILIC are able to detect certain polar metabolites. For a metabolomics discovery study, you generally have the best chance of capturing the full range of metabolites present in a sample if you select a multi-system approach that runs multiple detection instruments in tandem. 12 The 5 Key Elements of a Successful Metabolomics Study 11

13 Informatics: the Key to Good Data Regardless of the instrumentation used, a single metabolomics study is likely to identify hundreds of metabolites and produce thousands to tens of thousands of data points. Attempting to identify metabolites and derive biological significance from the mountain of data collected can be time-consuming and resourceintensive. Many metabolomics technologies use an ioncentric approach. This approach requires manual identification of all of the metabolites using ion features. This method has a high potential for false discovery and ambiguous identification, leading to substantially fewer accurately identified metabolites. On the other hand, an automated, chemocentric approach incorporates sophisticated software tools that distills the thousands of ion features, eliminate noise, and automatically classify metabolites by key ion features (mass, retention time and peak area). This approach requires an exhaustive chemical reference library of metabolite standards and sophisticated informatics software that can identify the metabolites. When this approach is used for broad, whole metabolome profiling, it ensures a more accurate measurement of the metabolites and their perturbation. It also allows for faster, easier and more accurate statistical analysis. The 5 Key Elements of a Successful Metabolomics Study 12

14 5. Have a Data Interpretation Plan in Place Nearly any metabolomics profiling technology will produce large amounts of complex data. You must have a plan in place for translating that data into meaningful biological insight. Your data interpretation plan should be driven by the deliverables you expect to receive from your metabolomics study. On the other hand, if your metabolomics study provider offers specialized interpretation tools or verified statistical analysis and pathway interpretation, your resources can be devoted to conducting any recommended follow-up studies and preparing your work for publication. If you will receive only the raw study data, be prepared to dedicate substantial time and resources to verifying the metabolite assignments, performing statistical analysis, and researching the biological implications of any metabolic perturbations. Be aware that this process can be time-consuming and challenging, particularly if your analyst does not provide you with specialized interpretation tools. Make sure to budget your resources accordingly. The 5 Key Elements of a Successful Metabolomics Study 13

15 Summary: The 5 Key Elements of Success To review, the five key elements of a successful metabolomics study are: 1. Define a Clear Objective 2. Use Strong Study Design Elements 3. Power Your Study for Success 4. Choose the Right Technology for Your Goals 5. Have a Data Interpretation Plan in Place When all of these components are present in your study, you dramatically improve your chances of deriving meaningful findings that translate into actionable results. Ready to speak to a metabolomics expert about your study? Click here. The 5 Key Elements of a Successful Metabolomics Study 14

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