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Metabolomics primarily focuses on the detection of various metabolites in samples, distinct from biomolecules such as nucleic acids and proteins. These metabolites have relatively small molecular masses, generally within 1000 Da, including amino acids, carboxylic acids, carbohydrates, alcohols, amines, lipids, and special substances like drugs and their degradation products.

Mass spectrometry, as a crucial tool in metabolomic research, has a history spanning over a century. In the late 19th century, E. Goldstein observed particles with positive charges through low-pressure discharge experiments. Subsequently, W. Wein found that positively charged particle beams could deflect under the influence of a magnetic field, laying the initial theoretical groundwork for mass spectrometry. In 1912, British physicist Joseph John Thomson developed the first simple mass spectrometer, and in 1919, Francis William Aston created the first precision mass spectrometer capable of measuring isotopes. The first commercial mass spectrometer was introduced in 1942.

In the 1950s, gas chromatography-mass spectrometry (GC-MS) was successfully developed, marking the integration of chromatography and mass spectrometry. In the 1960s, the invention of chemical ionization (CI) contributed to significant breakthroughs in mass spectrometry for detecting thermally unstable biomolecules. The 1970s witnessed the maturation of atmospheric pressure ionization (API) technology, resolving critical interface issues for liquid chromatography-mass spectrometry (LC-MS) and propelling LC-MS into widespread use. In the 1980s, the invention of "soft ionization" techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) enabled mass spectrometry to analyze non-volatile and highly polar substances.

At the turn of the century, the discipline of metabolomics officially emerged, transforming mass spectrometry from the study of individual metabolites to the analysis at the metabolome level. With the introduction of new concepts in metabolomics such as lipidomics, single-cell metabolomics, and spatial metabolomics, mass spectrometry plays an increasingly crucial role in metabolomic analysis.

Principle of Mass Spectrometer Identification

A mass spectrometer consists of several components, including the sample introduction system, ion source, mass analyzer, and detector. Since the operation of the mass spectrometer must take place in a vacuum environment, a specialized sample introduction system is required to allow samples at atmospheric pressure to enter the mass spectrometer without affecting the internal vacuum environment. Common sample introduction methods include direct injection, probe insertion, and chromatographic injection. For gases or volatile liquid pure compounds, such as some metabolite standards, they can generally be directly introduced into the ion source chamber of the mass spectrometer. For samples with relatively simple and less volatile components, a probe can be used to introduce them into the ion source chamber. The sample is then heated in the ion source until it vaporizes and undergoes ionization.

In cases where the sample composition is complex and less volatile, GC-MS or LC-MS is commonly used. The sample is first separated into relatively simple components using a chromatographic system before entering the mass spectrometer. This allows for simultaneous separation and mass spectrometric analysis during the process, or the separated components can be collected or combined before being sent for mass spectrometric analysis. Typically, biological samples such as urine, blood, cells, and tissue samples, which have complex metabolite compositions, are subjected to metabolomic analysis using gas chromatography or liquid chromatography coupled with mass spectrometry.

Sample Ionization

After the sample injection, the ion source ionizes the metabolite components, along with the solvent that has undergone chromatographic separation. There are various ionization methods, including electron impact ion source (EI), chemical ionization source (CI), fast atom bombardment ion source (FAB), electrospray ionization (ESI), atmospheric pressure chemical ionization source (APCI), laser desorption (LD), and others.

EI and CI are commonly used in GC-MS instruments. EI, as a hard ionization technique, typically utilizes a high voltage of 70 eV to collide the compound molecules with high-energy electrons. EI ionization is non-selective, ionizing most gaseous samples efficiently, and it produces abundant and fragmented ions. It is widely used in GC-MS applications. CI is divided into positive chemical ionization and negative chemical ionization. The former generates quasi-molecular ions, which is advantageous for relative molecular mass determination, while the latter is mainly used for identifying negatively charged compounds such as halogens, with limited application in routine metabolite detection.

Magnetic sector double-focusing mass spectrometers often use fast atom bombardment ionization for ionization, especially in trace analysis for preventive medicine and public health. LC-MS instruments commonly employ "soft ionization" methods such as ESI, APCI, and LD.

ESI, an atmospheric pressure ionization method, involves the formation of charged droplets when a sample solution passes through a high-voltage metal capillary (2-6 kV) due to electrostatic forces. After passing through a drying nitrogen gas curtain, the solvent rapidly evaporates, reducing the droplet surface area and increasing the surface charge density. This process repeats until Coulomb repulsion causes the droplet surface tension to reach the "Rayleigh limit," resulting in a "Coulomb explosion" that breaks the droplet into metabolite ions. ESI is widely used in metabolomic analysis due to its high ionization efficiency and cost-effectiveness.

Due to ESI's less effective ionization of small polar and non-polar molecules, APCI ionization can be used as a complementary method. APCI ionization operates at higher temperatures and employs chemical ionization to ensure the thorough ionization of such substances.

Matrix-assisted laser desorption/ionization (MALDI), based on laser desorption (LD) technology, involves coating the sample with a matrix. Upon high-energy laser heating, the matrix rapidly vaporizes, creating charged ions. Coating with a matrix helps evenly distribute laser energy to prevent compound destruction due to excessive energy. However, MALDI is more costly and has limited applications. Currently, MALDI mass spectrometry imaging combined with ion mobility is used for spatial metabolomic analysis in hot areas.

Mass Analysis of Ions

Metabolite molecules, after being ionized by the ion source, undergo detection of the ion's mass-to-charge ratio (m/z) by the mass analyzer, which is the core component of the entire mass spectrometer. In a vacuum environment, ions are deflected by a magnetic field, and ions with different m/z values exhibit different deflection patterns. Common mass analyzers, based on principles, include time-of-flight mass analyzer (TOF), quadrupole mass analyzer (Q), linear ion trap mass analyzer (LIT), Orbitrap mass analyzer (Orbitrap), and Fourier transform ion cyclotron resonance mass analyzer (FTICR). Based on resolution, Q and LIT belong to low-resolution mass analyzers, TOF belongs to medium-high-resolution mass analyzers, while Orbitrap and FTICR belong to high-resolution mass analyzers. In practice, different mass analyzers are often used in combination. For example, mass spectrometers from Agilent, AB Sciex, Thermo Fisher, and Waters are commonly used in metabolomics research, employing triple quadrupole (QQQ) technology and used for targeted metabolomics quantitative detection.

Models such as Agilent's 6495C, 6460C, 6470A, AB Sciex's Triple Quad 4500, Triple Quad 5500, Thermo Fisher's TSQ Fortis, TSQ Quantis, TSQ Altis, and Waters' Xevo TQ-S, Xevo TQD are based on triple quadrupole mass spectrometry technology and are commonly used for targeted metabolomics quantification. AB Sciex's Q-Trap series, including Q-Trap 4500, Q-Trap 5500, Q-Trap 7500, use quadrupole combined with linear ion trap mass spectrometry (Q-Trap) technology, providing 100 times higher resolution than conventional triple quadrupole technology. These are often used for widely-targeted metabolomics analysis. Quadrupole combined with time-of-flight mass spectrometry technology (Q-TOF), such as AB Sciex's TripleTOF 5600, TripleTOF 6600, and quadrupole combined with Orbitrap technology (Q-Orbitrap), such as Thermo Fisher's Q-Exactive, Q-Exactive Plus, Q-Exactive HF, Q-Exactive HFX, belong to high-resolution mass spectrometers, commonly used for untargeted metabolomics analysis. FTICR mass spectrometers have the highest resolution but are expensive and bulky, resulting in less widespread commercial use.

Ions detected by the mass analyzer are filtered based on m/z values by the quadrupole, usually prioritizing high abundance charged ions. Subsequently, ions with the same m/z value enter the collision cell, where they are fragmented by collisions with high-energy inert gas particles, often using Higher Energy Collision Induced Dissociation (HCD). This mode effectively breaks chemical bonds within the ions. The resulting ion fragments are then sent back to the mass analyzer for detection of their m/z values. Different types of ions yield different ion fragment information.

Detection of Ion Information

The detector converts ion beam information into electrical signals, amplifies the signals, and performs detection. During the analysis process, the instrument records information such as the m/z values and signal intensities of all ions and their respective fragments at a specific retention time. This information is stored as raw data files, serving as the offline data of the mass spectrometer. Subsequently, through data parsing and processing by analysis software, such as Thermo Fisher's Compound Discover, Lipidsearch, and open-source software like MS-Dial, and matching with annotation information from metabolite databases, a quantitative information table for the identification of all metabolites can be obtained. Commonly used analysis software applications allow for online connectivity to relevant metabolite databases for ion metabolite annotation.

Reference

1. Lokhov, Petr G., et al. "Mass spectrometry-based metabolomics analysis of obese patients' blood plasma." International Journal of Molecular Sciences 21.2 (2020): 568.