Inductively Coupled Plasma Mass Spectrometry (ICP-MS): Fundamentals and Analytical Applications

A key challenge in structural biology is to decipher molecular architectures and the interactions that regulate cellular functions. Techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron Microscopy (Cryo-EM) have greatly advanced our understanding of molecular structures, but often lack the resolution to analyze metal content, element-specific interactions, and the atomic distribution of heavy atoms in biomolecules. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a highly sensitive analytical tool that enables the precise detection of trace metals and isotopes.

Originally developed for environmental analysis, geochemistry, and clinical diagnostics, ICP-MS has become valuable in structural biology, particularly in the study of metalloproteins, metalloenzymes, and metal coordination within proteins. Since metals are essential for various biological functions - including enzyme activity, electron transfer, structural integrity, and signaling - understanding their interactions with biomolecules is critical to the study of cellular mechanisms.

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The Principles of ICP-MS

To understand the role of ICP-MS in structural biology, it is important to first understand the basic principles of the technique. ICP-MS combines the high-temperature capabilities of inductively coupled plasma (ICP) with the sensitive detection of mass spectrometry (MS) to identify and quantify elements at trace and ultra-trace levels. The ICP, which is typically formed by the interaction of an argon plasma with an electric coil to produce an electrically charged, ionized gas, atomizes and ionizes the sample, which is introduced into the plasma as an aerosol. The high-energy environment of the ICP ensures that the sample is efficiently atomized and ionized, converting it to charged ions.

Figure 1. The construction of Inductively Coupled Plasma torch. A: cooling gas tangential flow to the outer quartz tube. B: discharge gas flow (usually Ar). C: flow of carrier gas with sample. D: induction coil which forms the strong magnetic field inside the torch. E: force vectors of the magnetic field. F: the plasma torch (the discharge).

Once ionized, the sample passes through a series of mass spectrometer components: a quadrupole or sector field mass filter that separates ions based on their mass-to-charge ratio (m/z), followed by a detector that quantifies the ions. The intensity of the detected signal is proportional to the concentration of each element in the sample. ICP-MS offers exceptional sensitivity, with detection limits typically in the picogram to femtogram range, allowing the analysis of trace metals even in complex biological matrices such as cell lysates, tissues or even single cells.

Figure 2. Schematic diagram of the main components of an ICP-MS. (Mazarakioti et al., 2022)

One of the outstanding features of ICP-MS is its versatility in isotopic analysis. It can be used to measure both natural and isotopically enriched elements, allowing the study of metal incorporation, turnover and partitioning in biological systems. For example, isotopic labeling experiments can provide insight into the fate of specific metal ions within cellular processes, providing a window into the dynamic interactions between metals and biomolecules.

ICP-MS Trace Metals Analysis Workflow

Sample Preparation

Sample preparation is critical to ensure accurate and contamination-free results. Solid samples (e.g., soils, tissues) undergo acid digestion (e.g., HNO₃, HCl, or HF in microwave-assisted systems) to dissolve the matrix and release trace metals. Liquid samples (e.g., water, blood) may require filtration, acidification, or dilution to minimize matrix interferences. Internal standards (e.g., In, Sc, or Y) are added to correct for instrument drift and matrix effects. Rigorous labware cleaning and the use of high purity reagents (e.g., ≥18 MΩ-cm water) are essential to avoid contamination.

Sample Analysis

The prepared sample is introduced into the ICP-MS through a nebulizer, which converts it into an aerosol. The plasma (≥6000°C) ionizes the metals, which are then separated by a quadrupole mass analyzer based on their mass-to-charge ratio (m/z). Key parameters optimized include RF power, nebulizer gas flow, and collision/reaction cell settings to mitigate polyatomic interferences (e.g., ArO⁺ on ⁵⁶Fe). Calibration curves are generated using certified multi-element standards, and blanks are analyzed to subtract background signals. Quality control (QC) samples (e.g., spikes, certified reference materials) validate method accuracy and precision.

Data Analysis

Raw data (ion counts) are converted to concentrations using calibration curves. Internal standard ratios correct for signal suppression/enhancement. Data validation involves checking QC recovery (85–115%) and precision (RSD <10%). Advanced software tools (e.g., MassHunter, Qtegra) automate peak integration, interference correction, and uncertainty calculations. Results are reported with detection limits (ppt–ppb) and measurement uncertainties, ensuring compliance with regulatory standards (e.g., EPA, ISO).

Figure 3. ICP-MS trace metals analysis workflow.

Applications of ICP-MS Across Research Fields

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a versatile analytical technique extensively used across various research fields for its precision, sensitivity, and multi-element capabilities. Applications of ICP-MS across research fields are summarize in the figure 4 below.

• Environmental and Marine Sciences: ICP-MS is widely used in oceanography for multi-element bulk analysis, as exemplified by the GEOTRACES deep reference seawater samples. In this study, the instrument was used to analyze elemental elution profiles of dissolved iron at different oceanic baseline stations (Atlantic and Pacific), helping to investigate the spatial distribution and behavior of this critical element in oceanic waters. The profiles were collected during the GEOTRACES intercalibration cruises (2008 and 2009) and provide valuable data for the study of oceanic trace metal dynamics.
• Materials Sciences, Nanoscience and Technology: In environmental and materials science, ICP-MS aids in the temporal analysis of nanoparticles in soil samples. For example, in a study investigating the interaction of natural cerium- and lanthanum-containing particles (NNPs) with engineered nanoparticles (ENPs) such as CeO₂, ICP-MS was used to analyze mass spectra for individual nanoparticles. This research helps to track the fate and transport of engineered nanoparticles in the environment and provides insight into their potential impacts.
• Biology, Biochemistry, Pharmacology and Medicine: ICP-MS is also used in imaging mass cytometry for cancer research. In the analysis of HER2+ breast cancer tissue samples, ICP-MS is used to identify and map the presence of various proteins, such as cytokeratins, vimentin and HER2, within the tissue. The ability to overlay these biomarkers allows for detailed tissue profiling, supporting studies of cancer tissue heterogeneity and potential therapeutic targets. Imaging mass cytometry with ICP-MS enhances spatial resolution and aids in the understanding of protein interactions within tumors.
• Food Science Technology and Toxicology: ICP-MS plays a critical role in species-specific arsenic analysis, such as in rice samples from Rio Grande do Sul. Arsenic species in foods, such as rice, are accurately measured to assess their potential health risks. This application is critical to understanding the bioavailability and toxicity of arsenic, especially in regions where rice is a staple food and arsenic contamination is a concern.
• Geology, Geophysics and Geochemistry: In environmental geochemistry, ICP-MS is used for strontium isotope analysis to trace the sources of various geochemical processes. For example, ⁸⁷Sr/⁸⁶Sr ratios measured in Antarctic snow help to compare the isotopic composition of snow samples from different locations, such as Dome C and Vostok, and correlate them with potential source areas. This approach is essential for the study of climate change, ice core records, and geochemical cycles.

Figure 4. Applications of ICP-MS across research fields. (Van Acker et al., 2023)

Applications of ICP-MS in Structural Biology

Metalloproteins and Metalloenzymes: Key Targets for ICP-MS

One of the most compelling applications of ICP-MS in structural biology is the study of metalloproteins and metalloenzymes. These biomolecules, which incorporate metal ions as essential cofactors, are involved in a wide range of biochemical processes, including catalysis, electron transfer, and cellular regulation. The metal ions in metalloproteins often determine the three-dimensional structure and function of the protein, act as ligands for specific binding sites, and facilitate interactions with other molecules. The study of these proteins requires methods that can not only determine the identity of the metal ions involved, but also provide detailed information about their coordination and distribution within the protein structure.

ICP-MS is particularly adept at detecting metal content in metalloproteins, even at low concentrations. It allows precise quantification of metal ions such as zinc, copper, iron, manganese and magnesium, which are often present in trace amounts in biological systems. For example, in the case of cytochrome c oxidase, a key enzyme in the electron transport chain, ICP-MS can be used to measure iron and copper levels, providing insight into the enzyme's metal-dependent catalytic mechanism. In addition, ICP-MS can be used to identify metal incorporation and distribution in recombinant proteins produced in vitro, providing valuable information for optimizing protein expression conditions for structural studies.

In addition to detecting metals, ICP-MS can also help characterize metal-binding sites within proteins. Using isotopic labeling techniques, researchers can follow the incorporation of specific metal isotopes into the protein structure, providing valuable insight into the coordination environment and stability of metal centers. Such studies have been particularly useful in understanding the role of metals in enzymes involved in redox reactions, such as superoxide dismutase (SOD) and nitric oxide synthase (NOS), where metal cofactors are essential for catalysis.

Figure 5. ICP-MS-based approaches for in situ metallome imaging and metalloproteome identification. (A) Characterization of the metallomic profile in tissue sites. (B) General scheme of GE-ICP-MS for tracking metalloproteins. (Zhou et al., 2024)

Metal-Ion Coordination in Protein Structures

The coordination of metal ions within protein structures is a central aspect of metalloprotein function. The arrangement of ligands around a metal ion can influence the stability, reactivity, and specificity of the protein, and understanding these coordination environments is critical for elucidating the mechanistic details of metal-dependent enzymatic reactions. While techniques such as X-ray crystallography and NMR spectroscopy provide atomic-level information about protein structures, they often struggle to resolve the fine details of metal coordination, especially in cases where metals are bound to flexible or dynamic regions of the protein.

ICP-MS, in combination with other structural biology techniques, is a powerful tool for investigating metal coordination in proteins. By analyzing the metal content of protein samples with high precision, ICP-MS can help identify the stoichiometry of metal binding and determine the occupancy of metal sites. In addition, by using a variety of isotope-specific tracers, ICP-MS allows the study of metal incorporation into different metal-binding sites within a protein, providing a clearer picture of how metal ions are distributed within the protein structure.

Figure 6. Multifarious laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS)-based strategies have been developed and applied to investigate the metal-binding proteins in biospecimens in situ or through gel electrophoresis ex situ over the past decades, facilitating researchers disclosing how essential metals are implicated in life or what proteins toxic metals will target. (Chen et al., 2022)

For example, in the case of zinc fingers, a class of protein domains that bind zinc ions to stabilize their structural conformation, ICP-MS can be used to quantify the zinc content in the protein and track its incorporation into specific zinc-binding motifs. Similarly, in the study of metallothioneins, small cysteine-rich proteins involved in metal ion homeostasis, ICP-MS can provide valuable information about the binding of transition metals such as copper and zinc, which are crucial for the protein's function in metal detoxification and storage.

ICP-MS in Structural Characterization of Metal Complexes

Beyond metalloproteins, ICP-MS is also a powerful tool for studying metal complexes in structural biology. Metal ions often play a key role in the formation and stability of protein-ligand complexes, and understanding the interactions between metals and small molecules is essential for unraveling the mechanisms of metal-dependent signaling and catalysis. ICP-MS can be used to analyze the binding of metal ions to specific ligands, providing detailed information on the stoichiometry and affinity of metal-ligand interactions.

For example, ICP-MS has been used to study the interaction of metal ions with nucleic acids, such as the binding of magnesium ions to RNA, which is critical for RNA folding, stability and function. Similarly, ICP-MS can be used to study the binding of metals to synthetic ligands or drug molecules, providing insight into the potential for designing metal-based therapeutics. In the context of drug discovery, ICP-MS can be a valuable tool for screening metal-containing compounds and determining their metal-binding properties, which could guide the development of new therapies for diseases such as cancer, Alzheimer's and Parkinson's where metal dysregulation plays a key role in disease progression.

Figure 7. A heterogeneous bioassay for the detection of nucleic acids of HIV, HAV, and HBV was developed. The target DNA hybridized with both the immobilized capture strand and the metallic NPs. Above the DNA melting temperature, NPs were released and analyzed via SP ICP-MS. (Tian et al., 2022)

Advancements in ICP-MS for Structural Biology

The use of ICP-MS in structural biology has seen significant advances in recent years, particularly in the areas of sensitivity, resolution, and isotopic analysis. One of the most important improvements in ICP-MS technology has been the development of multi-collector systems that allow the simultaneous detection of multiple isotopes and elements. This capability enhances the ability to study metal interactions in complex biological systems, allowing researchers to explore the interplay between different metal ions and their role in cellular processes. For example, multi-collector ICP-MS can be used to study the interaction of copper and zinc in metalloproteins, shedding light on how these metals work together in enzymatic reactions or in stabilizing protein structures.

Another advance is the integration of ICP-MS with other complementary techniques, such as X-ray crystallography, NMR spectroscopy, and cryo-EM. This synergy allows researchers to combine the atomic-level structural information provided by these techniques with the metal-specific data obtained by ICP-MS, providing a more complete understanding of metal-dependent protein function. In particular, the combination of ICP-MS and cryo-EM holds great promise because it allows the study of metalloproteins in their native, non-crystalline state, providing new insights into the structural and functional roles of metals in dynamic biological systems.

Furthermore, the development of new sample preparation methods and improved instrumentation has enhanced the sensitivity and accuracy of ICP-MS, allowing for the analysis of even smaller sample volumes and the detection of trace metals in complex biological matrices. The ability to analyze single-cell samples is a particularly exciting development, as it opens up new possibilities for studying metal distribution and coordination in individual cells, which could have profound implications for understanding cellular metal homeostasis and metal-related diseases.

Figure 8. Perkin Elmer NexION 2000 ICP-MS.

By bridging the gap between metal analysis and molecular structure, ICP-MS is poised to play a critical role in unraveling the complexity of metal-containing biomolecules and their interactions in living systems. Whether used to characterize metal-binding sites in proteins, study metal ion dynamics in cellular processes, or explore the therapeutic potential of metal-based drugs, ICP-MS offers a powerful and versatile approach that complements traditional structural techniques and helps unlock the secrets of the molecular world.

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References

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3. Tian Y, Cheng J, Han X, et al. Novel thiol-functionalized covalent organic framework-enabled ICP-MS measurement of ultra-trace metals in complex matrices. J Anal at Spectrom. 2022;37(1):157-164.
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5. Zhou Y, Li H, Tse E, Sun H. Metal-detection based techniques and their applications in metallobiology. Chem Sci. 2024;15(27):10264-10280.