Flujo de trabajo del estudio metabolómico

Primer paso: diseño del estudio

Un resultado de investigación controlado y perspicaz depende en gran medida de la calidad de la preparación de su estudio. Antes de emprender cualquier estudio metabolómico, es de suma importancia diseñar bien el estudio, asegurándose de que las muestras que va a recoger y la forma de recogerlas son apropiadas y capaces de responder a su pregunta científica^.1 Planificar con antelación le ayudará a trazar su presupuesto y evitar la necesidad inesperada de añadir más muestras más adelante o solicitar ampliaciones de financiación. También es importante eliminar, o al menos identificar, las variables externas que puedan afectar a su estudio, de modo que pueda tenerlas en cuenta posteriormente durante el análisis de los datos.

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.).

Segundo paso: preparación de la muestra

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 le facilita la recogida y el almacenamiento de muestras mediante nuestro kit gratuito de manipulación de muestras Study Success, personalizado para su estudio. En el kit se incluyen tubos con códigos de barras específicos para su matriz (es decir, biofluidos, muestras sólidas o pellets celulares), un escáner de códigos de barras e instrucciones para enviarnos sus muestras por correo para garantizar que lleguen de forma segura. Si almacena grandes cantidades de muestras en un biorrepositorio o biobanco, trabajamos directamente con su personal para preparar y enviar sus muestras.

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.

También puede estar seguro de que sus datos se ajustarán a las normas de calidad más exigentes: nuestros laboratorios cuentan con la certificación ISO 9001:2015 para pruebas analíticas y diagnósticas de muestras biológicas y con la certificación CLIA y la acreditación del Colegio de Patólogos Americanos (CAP) para pruebas diagnósticas de muestras humanas.

Tercera etapa: Análisis de muestras

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.

Mediante la espectrometría de masas, las pequeñas moléculas presentes en las muestras se ionizan en una fuente de iones y, a continuación, se detectan en un detector de masas acoplado. Estas pequeñas moléculas pueden introducirse en el espectrómetro de masas mediante infusión directa o, más comúnmente, a través de un sistema cromatográfico acoplado^.1 La cromatografía de gases (GC) y la cromatografía líquida (LC) se han utilizado durante años para analizar pequeñas moléculas; sin embargo, la LC-MS está ganando popularidad porque puede identificar y medir una gama más amplia de compuestos que la GC-MS, y la preparación de muestras es más sencilla^.5

La plataforma de metabolómica LC-MS patentada de Metabolonestá preparada para detectar más metabolitos únicos que cualquier otra plataforma disponible, lo que garantiza que pueda extraer el máximo conocimiento biológico posible de sus muestras. También disponemos de una gama de paneles optimizados, dirigidos y no dirigidos, y ensayos de un solo analito que cubren miles de metabolitos y lípidos en una amplia gama de clases bioquímicas, rutas metabólicas y procesos fisiológicos. Nuestros paneles dirigidos cuentan con métodos precisos y totalmente validados para una cuantificación absoluta. Y si no tenemos un ensayo que pueda responder a su pregunta de investigación, nuestros científicos expertos trabajarán con usted para desarrollar un ensayo personalizado para sus necesidades.

Cuarto paso: Adquisición, tratamiento, análisis e interpretación de datos

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.

¿Y ahora qué?

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.

Referencias

1. https://www.ebi.ac.uk/training/online/courses/metabolomics-introduction/designing-a-metabolomics-study/design-experiment/
2. https://www.metabolon.com/working-with-us/study-design-consulting/
3. https://www.metabolon.com/working-with-us/sample-handling-kit/
4. Marshall DD and Powers R. Beyond the Paradigm: Combining Mass Spectrometry and Nuclear Magnetic Resonance for Metabolomics. Prog Nucl Magn Reson Spectrosc 2017; 100: 1–16. doi: 10.1016/j.pnmrs.2017.01.001
5. Perez ER, Knapp JA, Horn CK et al. Comparison of LC–MS-MS and GC–MS Analysis of Benzodiazepine Compounds Included in the Drug Demand Reduction Urinalysis Program. J Anal Toxicol 2016; 40(3): 201–207. doi: 10.1093/jat/bkv140
6. https://www.metabolon.com/services/discover/
7. https://www.metabolon.com/services/target/
8. https://www.metabolon.com/services/target/#singleanalyte
9. https://www.metabolon.com/resources/webinars/client-data-table/
10. https://www.metabolon.com/resources/videos/mymetabolon-portal/
11. https://www.metabolon.com/working-with-us/reporting-interpretation/
12. https://www.metabolon.com/publications