Mass Spectrometry for Metabolomics: Techniques, Applications, and Instrumentation

Metabolomics primarily targets the various metabolites present in samples, which differ from larger biomolecules like nucleic acids and proteins. These metabolites typically have a relatively small molecular weight, generally below 1000 Da. They include a wide array of substances such as amino acids, carboxylic acids, carbohydrates, alcohols, amines, and lipids. Additionally, metabolomics can also detect specific compounds like pharmaceuticals and their metabolites.

Mass spectrometry has been a pivotal tool in metabolomics research, with a history that spans over a century. In the late 19th century, E. Goldstein first observed particles with a positive charge during low-pressure discharge experiments. Following this discovery, W. Wein demonstrated that these positively charged particles could be deflected by a magnetic field, laying the foundational theory for mass spectrometry. In 1912, British physicist Joseph John Thomson developed the first rudimentary mass spectrometer. By 1919, Francis William Aston had successfully created a precision mass spectrometer capable of isotopic determination.

The commercial introduction of mass spectrometers occurred in 1942. The 1950s saw the advent of the Gas Chromatography-Mass Spectrometry (GC-MS), a significant milestone marking the integration of chromatographic techniques with mass spectrometry. The invention of Chemical Ionization (CI) in the 1960s represented a significant breakthrough in the analysis of thermally unstable biomolecules. During the 1970s, the development of Atmospheric Pressure Ionization (API) technologies addressed key interfacing challenges in Liquid Chromatography-Mass Spectrometry (LC-MS), propelling it into widespread use.

In the 1980s, the invention of "soft ionization" techniques such as Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI) allowed for the analysis of non-volatile and highly polar substances. The turn of the century marked the formal emergence of metabolomics as a discipline, shifting the focus of mass spectrometry from the study of individual metabolites to the comprehensive analysis of metabolomes.

Recent developments have introduced new concepts such as lipidomics, single-cell metabolomics, and spatial metabolomics, further emphasizing the crucial role of mass spectrometry in metabolomic analysis. These advancements have significantly broadened the scope and application of mass spectrometry in the study of complex biological systems.

Principle of Mass Spectrometer Identification

Mass spectrometers are complex instruments that consist of several key components: a sample introduction system, an ion source, a mass analyzer, and a detector. The operation of a mass spectrometer requires a vacuum environment to prevent air pressure from interfering with the internal mechanisms. Specialized sample introduction systems are designed to maintain this vacuum while allowing samples to enter the spectrometer.

Common methods of sample introduction include direct insertion, probe insertion, and chromatographic introduction. For gases or volatile liquid compounds, such as certain metabolic standards, samples can be directly introduced into the ion source chamber of the mass spectrometer. For samples that are simple in composition and less volatile, a probe can be used to introduce the sample into the ion source chamber. The sample is then heated until it vaporizes, at which point ionization occurs.

More complex samples typically require the use of chromatography coupled with mass spectrometry. The sample first passes through a chromatographic system, which separates it into simpler components. These components can then be continuously analyzed by the mass spectrometer as they emerge from the chromatographic system, or they can be collected and then introduced to the spectrometer after separation.

Biological samples such as urine, blood, cells, and tissue samples, which contain highly complex mixtures of metabolites, generally utilize either gas chromatography (GC) or liquid chromatography (LC) in tandem with mass spectrometry. This combination allows for the detailed analysis of metabolomes and is crucial for metabolic profiling in metabolomics studies.

Simplified diagram of a mass spectrometer (Rankin et al., 2014).

Sample Ionization

After sample introduction, the ion source in a mass spectrometer ionizes the metabolite components separated by chromatography, along with the solvent. There are various ionization methods available, including electron impact (EI), chemical ionization (CI), fast atom bombardment (FAB), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and laser desorption (LD).

EI and CI are commonly used in GC-MS systems. EI, known as a "hard ionization" technique, typically uses high voltage (about 70eV) to induce ionization by colliding molecules with high-energy electrons. EI is non-selective, capable of ionizing general vaporized samples with high ionization efficiency and generating rich fragment ions, making it the most widely used method in GC-MS applications. CI can be in the form of positive or negative ion modes; the former generates quasi-molecular ions which are beneficial for molecular weight determination, while the latter is mainly used for negatively charged compounds like halogens, less frequently used in standard metabolite analysis.

FAB ionization, typically used in magnetic sector mass spectrometers, is often employed in trace analysis within fields like preventive medicine and public health. In LC-MS systems, "soft ionization" techniques such as ESI, APCI, and LD are commonly employed. ESI operates under atmospheric pressure where the sample solution passes through a high-voltage metal capillary (2-6 kV), creating a charged mist through electrostatic effects. This mist is then dried by a stream of nitrogen, causing the solvent to evaporate rapidly and the surface area of the droplets to decrease, thereby increasing the surface charge density until Coulombic repulsion causes the droplets to explode in a process known as "Coulomb explosion." This process repeats until solvent-free metabolic ions are formed. ESI is the most widely used due to its high ionization efficiency and cost-effectiveness in metabolomics.

However, ESI is less effective at ionizing small, non-polar molecules, making complete ionization challenging. APCI, on the other hand, can fully ionize such substances at higher temperatures through chemical ionization, often serving as a complementary method to ESI. Matrix-assisted laser desorption/ionization (MALDI) builds on the LD technique by embedding the sample in a matrix that is co-vaporized with it upon high-energy laser irradiation, creating charged ions. This method prevents compound degradation by dispersing the laser energy evenly but is more costly and less frequently used. Currently, MALDI imaging technology combined with ion mobility is gaining traction for spatial metabolomics analysis.

Classification of Ionization Sources Commonly Used in Mass Spectrometers (Gerardi et al., 2009).

Mass Analysis of Ions

After ionization in the ion source, metabolite molecules in a mass spectrometer are detected by the mass analyzer, the core component of the instrument. In a vacuum environment, ions are deflected by a magnetic field based on their mass-to-charge ratio (m/z). Common mass analyzers include time-of-flight (TOF), quadrupole (Q), linear ion trap (LIT), Orbitrap, and Fourier transform ion cyclotron resonance (FTICR) analyzers.

Based on resolution, Q and LIT are low-resolution mass analyzers, TOF is a medium-to-high-resolution analyzer, and Orbitrap and FTICR are high-resolution analyzers. Typically, different mass analyzers are combined for specific applications. For example, instruments from Agilent, AB Sciex, Thermo Fisher, and Waters often employ triple quadrupole (QQQ) technology for targeted metabolomics quantification. The Q-Trap series from AB Sciex utilizes a combination of quadrupole and linear ion trap technology, providing 100 times higher resolution than conventional QQQ instruments, commonly used for widely-targeted metabolomics analysis. Instruments like Q-TOF (e.g., AB Sciex TripleTOF series) and Q-Orbitrap (e.g., Thermo Fisher Q-Exactive series) offer high resolution for untargeted metabolomics studies.

Following detection, ions are filtered by the quadrupole based on their m/z values, with priority given to high-abundance ions. Subsequently, ions with the same m/z values are subjected to fragmentation in the collision cell by collision-induced dissociation (CID), often employing higher energy collision-induced dissociation (HCD). This fragmentation opens chemical bonds within the ions, and the resulting fragments are then re-analyzed by the mass analyzer to determine their m/z values. Different types of ions produce distinct fragmentation patterns, providing valuable information for compound identification.

Detection of Ion Information

The detector converts the ion beam information into electrical signals, amplifies them, and detects them. During analysis, the instrument records all ions and their respective fragment m/z values and signal intensities at a specific retention time. This data, known as raw files, serves as the basis for subsequent analysis. Using data parsing algorithms in analysis software and matching annotation information from metabolite databases, quantitative information for identified metabolites can be obtained. Commonly used analysis software includes Thermo Fisher's Compound Discover, LipidSearch, as well as open-source software like MS-Dial. These software platforms often integrate with relevant metabolite databases for metabolite annotation.

Commonly Used Mass Spectrometers for Metabolomics

Thermo Fisher Scientific

Model: Orbitrap Fusion Lumos

• Description: The Orbitrap Fusion Lumos is a state-of-the-art mass spectrometer that offers exceptional sensitivity and resolution. It is equipped with a Tribrid architecture that combines quadrupole, Orbitrap, and ion trap mass analyzers. This flexibility allows for a wide range of experiments, from routine quantification to in-depth structural elucidation.
• Applications: Ideal for complex metabolomic profiling due to its high-resolution accurate mass (HRAM) capabilities and its ability to handle multiple fragmentation techniques (CID, HCD, and ETD).

Model: Q Exactive HF

• Description: This model is highly popular in metabolomics for its high-performance quadrupole-Orbitrap mass analyzer, providing high accuracy, resolution (up to 240,000 at m/z 200), and scan speed. It simplifies quantitation and identification workflows while maintaining high throughput.
• Applications: Suitable for targeted and untargeted metabolomics due to its robustness and ease of integration with UHPLC systems.

Agilent Technologies

Model: Agilent 6550 iFunnel Q-TOF

• Description: The 6550 Q-TOF features Agilent's unique iFunnel technology, which significantly boosts sensitivity. It offers high resolution and accurate mass capabilities, making it a powerful tool for complex sample analysis in metabolomics.
• Applications: Excellent for both qualitative and quantitative analyses, and highly effective for discovering new metabolites.

Waters Corporation

Model: SYNAPT G2-Si

• Description: The SYNAPT G2-Si is a high-definition mass spectrometer that combines quadrupole time-of-flight (Q-TOF) technology with Waters' unique ion mobility separation technology. This combination enhances the resolution of complex mixtures and provides another dimension of separation.
• Applications: Particularly useful in metabolomics for distinguishing structurally similar metabolites and for in-depth lipidomics.

SCIEX

Model: TripleTOF 6600

• Description: The TripleTOF 6600 offers high-resolution TOF/MS and MS/MS capabilities, making it ideal for large-scale metabolomic studies. It provides exceptional speed and sensitivity, facilitating rapid data acquisition with minimal sample consumption.
• Applications: Well-suited for both targeted and exploratory metabolomics. Its high-throughput capabilities make it excellent for biomarker discovery.

Bruker Corporation

Model: timsTOF Pro

• Description: Incorporating Bruker's proprietary trapped ion mobility spectrometry (TIMS) along with Q-TOF mass spectrometry, the timsTOF Pro excels in separating and identifying metabolites in highly complex samples.
• Applications: Particularly effective in lipidomics and small molecule imaging. Its high sensitivity and resolution improve confidence in metabolite identification.

References

1. Rankin, Naomi J., et al. "The emergence of proton nuclear magnetic resonance metabolomics in the cardiovascular arena as viewed from a clinical perspective." Atherosclerosis 237.1 (2014): 287-300.
2. Gerardi, Anthony R., Jennifer L. Lubbeck, and Christa L. Colyer. "Dimethylditetradecylammonium bromide (2C 14 DAB) as a self-assembled surfactant coating for detection of protein–dye complexes by CE-LIF." Journal of Solid State Electrochemistry 13 (2009): 633-638.