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Study Design

Chapter 2 — Metabolomics Study Workflow

Now that we’ve reviewed some of the most important considerations to make when deciding who to work with for your metabolomics studies (if you will not perform your studies in-house), it’s time to dive deep into the metabolomics study workflow. In this chapter, we provide an overview of each step in the workflow, from study design to data analysis and interpretation, and how Metabolon can help you each step of the way.

Step One: Study Design

A controlled and insightful research outcome is highly dependent on the quality of your study preparation. Before you undergo any metabolomics study, it is of utmost importance to design your study well, ensuring that the samples you will collect and how you collect them are appropriate and capable of answering your scientific question.1 Planning ahead will help you map out your budget and avoid the unexpected need to add more samples later or request funding extensions. It is also important to eliminate or at the very least, identify, external variables that can impact your study so you can take them into account later during data analysis.

At Metabolon, we work with you to ensure three key components of a successful study: a clearly defined objective, strong study-design elements, and adequate study power. Every study we do begins with a one-on-one consultation to fully understand your research goals. Then we work with you to design a metabolomic study with proper consideration of sample types, time points, doses, and phenotypic/disease severity. We help minimize variation and noise by identifying the ideal sample matrix for your study, ensuring the appropriate controls in your study design, and making sure your study collects adequate exposure data (ie, dose, time of collection, etc.).

Step Two: Sample Preparation

Sample preparation includes several steps that must be carefully considered and carried out to ensure you will produce high-quality data from your samples: collection, storage, extraction, and preparation for measurement with specific instrumentation.1

As we’ll discuss in more detail in Chapter 4 of this guide, there are many different sample types that can be analyzed using metabolomics protocols, including but not limited to cells in culture, tissue, feces, blood, urine, and sweat. The sample source you use depends upon your research goal; for example, you might select stool if you want to study the impact of the gut microbiome on host nutrient acquisition, tissue samples if you want to identify biomarkers that differentiate primary tumors and metastases, or plasma if you are looking at systemic changes. Each sample type requires a unique method of handling and preparation, which is influenced by whether you’ve selected a targeted or untargeted approach for your metabolomics profiling study.

Metabolon makes sample collection and storage easy for you through our free Study Success Sample Handling Kit, customized for your study. Included in the kit are barcoded tubes specific to your matrix (i.e., biofluids, solid samples, or cell pellets), a barcode scanner, and instructions for mailing your samples to us to ensure they arrive safely. If you store large quantities of samples in a biorepository or biobank, we work directly with personnel there to prepare and ship your samples.

Once your samples arrive, we store them in carefully monitored, -80 °C freezers backed by generators or in other sample-appropriate storage until they are processed. We have the knowledge and expertise to ensure that your samples, no matter their source, are prepared for analysis appropriately, whether your study utilizes a global, untargeted approach, a targeted panel, or a combination of the two.

You can also be sure that your data will adhere to the highest quality standards: our labs have ISO 9001:2015 certification for analytical and diagnostic testing of biological specimens and are CLIA certified and accredited by the College of American Pathologists (CAP) for diagnostic testing on human specimens.

Step Three: Sample Analysis

Once your samples are processed, the next step is to extract information about the metabolites and metabolic pathways present in the samples using the appropriate instrumentation for your study design. As we described in Chapter 1 of this guide, there are two main approaches for analyzing metabolites: nuclear magnetic resonance (NMR) and mass spectrometry (MS). Because MS is more sensitive than NMR,1,4 it is often the tool of choice for metabolomics studies.

Using mass spectrometry, small molecules present in your samples are ionized at an ion source and then detected in a coupled mass detector. These small molecules may either be introduced into the mass spectrometer via direct infusion or, more commonly, through a coupled chromatographic system.1 Gas chromatography (GC) and liquid chromatography (LC) mass spectrometry have both been used for years to analyze small molecules; however, LC-MS is growing in popularity because it can identify and measure a broader range of compounds than GC-MS, and sample preparation is more straightforward.5

Metabolon’s proprietary LC-MS metabolomics platform is powered to detect more unique metabolites than any other platform available, ensuring you can extract as much biological insight from your samples as possible. We also have a range of optimized untargeted and targeted panels and single analyte assays covering thousands of metabolites and lipids across a broad range of biochemical classes, metabolic pathways, and physiological processes. Our targeted panels boast precise and fully validated methods for absolute quantification. And if we don’t have an assay that can answer your research question, our expert scientists will work with you to develop a custom assay for your needs.

Step Four: Data Acquisition, Processing, Analysis, and Interpretation

The output of mass spectrometers is typically a matrix (i.e., a large table full of rows and columns) listing the peaks/signals that represent the compounds present in your sample. They are reported as m/z, or the mass to charge of each compound.1 The key is to extract meaningful biological insights from this table, which can be a daunting task for anyone who is unfamiliar with mass spectrometry data outputs and the tools that can be used to analyze them. Correcting for and/or removing noise is necessary for data analysis and subsequently, accurate interpretation leading to actionable insights.

Metabolon’s team of experts has decades of experience in analyzing and interpreting LC-MS data. While Metabolon does deliver every customer a formatted Client Data Table that is ready for import and analysis with third-party tools such as R and Python, Global Discovery panels additionally come with continuous access to the MyMetabolon portal, where you can find customized interpretation by our PhD-level study directors, statistical tests they performed, fully customizable data visualization tools, a comprehensive summary of actionable insights, and recommended next steps. Additionally, the Complex Lipid Targeted panel comes with access to the Surveyor data visualization tool.

We often work with customers after initial data interpretation and delivery to help them analyze their datasets more deeply and to design and execute follow-up studies. These collaborative efforts have led to the publication of over 3,000 scientific presentations and publications. These collaborations are fruitful because when you work with Metabolon, you’re not working with a fee-for-service vendor; you’re working with a team of PhD-trained scientists eager to help you uncover exciting new scientific discoveries.

What’s Next?

In this Chapter, we discussed some of the key components of a successful metabolomics study workflow, including key considerations for study design, sample preparation, as well as data analysis and interpretation. In the next chapter of this guide, we’ll take a deep dive into the nuts and bolts behind designing a metabolomics study, which includes how to define your study’s purpose, selecting an appropriate experimental approach, determining the size of your study, selecting controls, ensuring your study is appropriately powered, and setting appropriate expectations from your results.

metabolomics study design success guide

Continue to Chapter 3 - Building Your Metabolomic Study

There are a number of factors to consider when designing your study to ensure your protocol delivers high-quality results for drawing actionable insights. In this chapter, we’ll go over each of these factors, breaking the process down into simple steps with clear, easy-to-follow advice.

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