Mass Spectrometry-Based Metabolomics

Mass spectrometry is one of the most widely used techniques for metabolomics, the study of metabolites. Biocompare recently consulted experts from leading companies—SCIEX, Thermo Fisher Scientific, and Waters Corporation—to gather their insights and best practices for optimizing mass spectrometry workflows in metabolomics.

Avoiding contamination at the earliest stages

Rebekah Sayers, Ph.D., Manager of Small Molecule Omics at SCIEX, stresses that metabolites are often highly unstable and prone to degradation during sample collection, storage, and preparation. She also warns that temperature, pH, and enzymatic activity can alter metabolite levels. “Proper sample preparation and extraction are critical for obtaining accurate and reproducible results in any mass spectrometry-based analysis,” she emphasizes.

Dr. Sayers argues for consistency in sample handling and storage to minimize variability and prevent degradation. “Taking the time to aliquot samples and limiting freeze-thaw cycles and thawing on ice will improve the reliability and reproducibility of your data.”

She also advises that researchers avoid contamination by using clean labware and high-purity solvents and reagents. “Do not reuse pipette tips or tubes, be aware of how your glassware is cleaned, avoid detergents, and thoroughly rinse with solvent before use.”

Tips for extraction

Dr. Adam King, Ph.D., Senior Scientist at Waters Corporation, provides advice for choosing an extraction method—such as liquid-liquid extraction, solid-phase extraction, and protein precipitation. He recommends that researchers begin by identifying the type of analysis to be performed.

Key article takeaways

1. Minimize contamination and sample degradation: Proper sample handling is crucial to avoid contamination and preserve metabolite stability. Experts stress the importance of using clean labware, high-purity solvents, and proper storage methods (e.g., aliquoting samples and avoiding freeze-thaw cycles) to ensure reproducibility and accuracy in metabolomics analyses.

2. Choosing the right extraction method: The choice of extraction technique (e.g., liquid-liquid extraction, solid-phase extraction, or protein precipitation) depends on the type of analysis being performed. For untargeted metabolomics, simpler extraction methods are preferred to minimize analyte loss. For targeted analyses, methods like solid-phase extraction can optimize recovery of specific metabolites while removing unwanted compounds.

3. Optimizing sample analysis with complementary chromatography: Using complementary chromatography techniques (e.g., reversed-phase chromatography and hydrophilic interaction chromatography, or HILIC) can improve metabolite coverage. System suitability checks should be performed regularly to monitor instrument performance and ensure data quality, especially when using HILIC or other sensitive methods.

4. Selecting the right acquisition Mode (DDA vs. DIA): The choice between DDA and DIA depends on the study's goals. DDA is ideal for discovery-based research where high-quality MS/MS spectra of abundant ions are required, while DIA offers more comprehensive coverage and is suited for large-scale, quantitative studies, though it demands more sophisticated bioinformatics tools.

Dr. King notes that mass spectrometry-based metabolomics studies can be broadly categorized into two approaches: targeted and untargeted (or discovery) methods. Targeted metabolomics analyzes a predefined set of known metabolites, while untargeted metabolomics seeks to measure all detectable metabolites in a sample, including unknowns.

For untargeted research, Dr. King advises minimizing the steps during sample preparation and extraction. He warns that complex extraction methods with many steps increase the risk of losing analytes. “Therefore, simplicity is key: remove proteins via precipitation, centrifuge, dry down supernatant, and reconstitute in a diluent appropriate to the chromatographic method being used.”

In contrast, in the case of targeted analysis, he stresses how the physicochemical properties of the molecules of interest can be leveraged to optimize their recovery from the matrix—while removing interfering molecules that are not of interest. For targeted analysis, he recommends solid-phase extraction, including the Waters Oasis solid-phase extraction products.

Dr. Sayers further advises that researchers use internal standards for the extraction process. “By adding internal standards at the beginning of the extraction process, you can account for variability in extraction efficiency.” She also recommends using blanks during extractions to help identify potential contaminants introduced during the process.

Tips for running samples

Dr. King suggests that researchers analyze samples with complementary chromatography techniques—such as reversed-phase chromatography and hydrophilic interaction chromatography (HILIC)—to increase coverage and ensure comprehensive detection of metabolites. “While using complementary techniques will increase the overall analysis time for a single study sample, the resulting data is far richer and provides for more information.”

He also recommends that researchers perform system suitability injections before, during, and at the end of the analysis using well-characterized standards. “This allows you to monitor the performance of your system. Although modern instruments are very robust, it is always best practice to check analyte retention time, response, and mass accuracy before analyzing your precious study sample.”

For HILIC analysis, Dr. King recommends the new Waters Atlantis™ Premier BEH Z-HILIC column for its specificity. Meanwhile, Dr. Sayers advises that samples for HILIC analysis be prepared in a solvent composition compatible with the HILIC mobile phase. “This is typically a high organic content similar to the initial conditions of the gradient.”

Finally, Dr. Sayers stresses that injection volumes should be kept low to avoid overloading and ensure consistent peak shapes. “This is essential when running HILIC to maintain the stability of the retention mechanism.”

Furthermore, she argues that smaller injection volumes can improve the signal-to-noise ratio for mass spectrometry detection. “Overloading the column can lead to matrix effects and ion suppression in mass spectrometry, reducing sensitivity and accuracy. Smaller volumes help maintain cleaner spectra and more reliable quantitation,” she adds.

Tips on selecting the acquisition mode

Dr. Sayers explains that both data-dependent acquisition (DDA) and data-independent acquisition (DIA) approaches can be used for metabolomics studies.

In DDA, only the most abundant ions are typically chosen for subsequent MS/MS analysis in each cycle. “By focusing on the most intense ions, DDA provides high-quality MS/MS spectra for those selected ions, which is very effective for identifying unknown metabolites.” However, less abundant ions may also be missed.

In contrast, with DIA, the mass spectrometer fragments all ions within a specified m/z range, independent of their intensity. “This can provide more comprehensive coverage, but the MS/MS spectra may be less clear due to the simultaneous fragmentation of multiple ions, leading to more complex spectra.” DIA methods also tend to require sophisticated bioinformatics tools for processing and interpretation.

“The choice between DDA and DIA depends on the specific goals and constraints of the metabolomics study,” explains Dr. Sayers. “DDA is suitable for discovery-driven research where high-quality MS/MS spectra of the most abundant ions are needed. It is also easier to implement and requires less computational power.”

On the other hand, she argues that DIA is ideal for comprehensive profiling and quantitative studies where consistent coverage and reproducibility are critical. “DIA is particularly useful for large-scale studies that require robust quantitation across many samples.”

Dr. King, who worked with the National Phenome Centre in London, agrees and highlights the Waters approach to DIA. “Waters DIA approaches, such as MS^E, HDMS^E, and SONAR, have been utilized for a number of years to balance the collection of quantifiable, high-sensitivity mass spectrometry data with high-resolution, accurate mass fragment ion information in large-cohort studies.”

Finally, Dr. Sayers argues that metabolomics researchers consider combining targeted and untargeted approaches to achieve a more comprehensive and reliable metabolic profile.

New workflows and instrumentation for metabolomics

At the 2024 America Society of Mass Spectrometry conference, Waters unveiled the Xevo MRT Mass Spectrometer, a high-performance, quadrupole time-of-flight instrument designed for metabolomic workflows.

The platform’s new multi-reflecting time-of-flight (MRT) mass analyzer combines high resolution (100,000 FWHM) and high mass accuracy (< 500 ppb) with fast acquisition rates, maximizing high-quality data collection from the faster chromatographic methods necessary for large population and epidemiological studies. The instrument was introduced alongside new informatics workflows compatible with many leading metabolomics software platforms. This allows for easy integration into a lab’s existing data pipeline.

Dr. Sayers highlights that the novel Zeno trap from SCIEX significantly increases signal intensity and improves detection limits. This capability is particularly beneficial in metabolomics, where detecting low-abundance metabolites can provide deeper insights into metabolic pathways and disease mechanisms. In addition, EAD fragmentation technology features a novel tunable EAD device, which enables the analysis of both singly charged precursors, such as lipids and metabolites, and multiply charged precursors, such as peptides. Electron-activated workflows are greatly impacting metabolomics by facilitating the separation of isobaric compounds and the precise determination of their molecular structures.

Susan Bird, Manager of Vertical Marketing at Thermo Fisher Scientific, emphasizes how the company has developed simultaneous quantitation and discovery (SQUAD) metabolomics, allowing researchers to combine targeted and untargeted workflows. She notes that Orbitrap-based mass spectrometers offer several advantages to optimize this process, such as fast polarity switching to conduct positive and negative mode analyses in a single injection while still obtaining good quantitation.

She also notes how recent advances in mass spectrometry—such as the Thermo Scientific Orbitrap Tribrid mass spectrometer and the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer—feature instruments with two detectors that can operate in parallel, allowing researchers to run targeted and untargeted experiments simultaneously.

“This setup provides the robustness of untargeted experiments while retaining the precision of targeted ones. This enables metabolomics researchers to utilize data more effectively, akin to how proteomics and other omics data are leveraged.”

Finally, Bird highlights how the Thermo Scientific™ AcquireX™ intelligent data acquisition workflow generates comprehensive fragmentation coverage of unique sample-relevant compounds by the automated exclusion of non-biological and redundant features. This provides improved identification of unknowns and higher-quality annotations.