Multimodal Spectrometry Tag Enhances Protein Detection in Tissue Imaging

Detecting very small amounts of proteins in tissue is tricky with standard staining methods. One way scientists boost the signal is with something called tyramide signal amplification (TSA), which uses an enzyme to create highly reactive molecules that quickly stick right where the target protein is, giving a stronger and more precise signal. This method works across several imaging techniques and can be tweaked in different ways to increase sensitivity. More recently, researchers have explored mass spectrometry imaging as an alternative, which uses metal-tagged antibodies to map proteins with high sensitivity and the ability to measure multiple targets at once, though it can be slower and damage the sample. To combine the strengths of both approaches, scientists are developing special tags, like a newly designed ruthenium-based molecule, that can be seen with both fluorescence and mass spectrometry. This allows researchers to first locate areas of interest using fluorescence, then analyze them in more detail with mass spectrometry, improving detection of low-level proteins.

A recent paper published in Chemical & Biomedical Engineering¹ reported on the synthesis of a bespoke ruthenium complex (TyrRu) for tyramide amplification and describe its application as a multimodal analyte to image and quantify glial fibrillary acidic protein in mouse brain by complementary immunofluorescence and elemental mass spectrometry imaging (MSI). LCGC International spoke to David Bishop, corresponding author of that paper, about the work.

How does coupling tyramide signal amplification with elemental mass spectrometry imaging improve the detection of low-abundance proteins compared with traditional immunofluorescence methods?

Tyramide signal amplification and elemental mass spectrometry do not improve detection over traditional immunofluorescence methods that also use tyramide signal amplification with brighter fluorophores. The complex, TyrRu, that we synthesized here showed improved detection via elemental mass spectrometry imaging (MSI), as indicated by the signal to noise, but it is a weaker fluorescent dye compared to commercially available fluorescing agents.

What advantages does laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) offer for quantitative tissue imaging compared with optical imaging techniques?

Understanding how cells function requires knowledge on the location and number of biomolecules such as proteins and mRNA. Optical imaging techniques are useful for imaging biomolecules in tissues, however they are qualitative and it is typically difficult to simultaneously identify multiple biomolecules. Additionally, a second complementary quantification measurement is required. In comparison, LA-ICP-MS can spatially quantify multiple biomolecules directly on tissue sections when analyzed alongside external matrix-matched quantification standards. LA-ICP-MS is however slower, and an ICP-time-of-flight (TOF) is required to analyze larger numbers of analytes simultaneously.

Why are metal-conjugated probes particularly well suited for mass spectrometry imaging applications in biological tissues?

In a similar manner to immunohistochemistry, we use probes such as antibodies to specifically recognize the target biomolecules. However, we replace the chromogenic or fluorescent visualization reagent with metal analytes that are required for detection by elemental MSI. We typically select metals that are not endogenous and are easily ionized as analytes. Here, as we wanted to synthesize a multimodal reagent, we chose ruthenium.

For more information on the advantages of elemental MSI and metal-conjugated probes for analyzing biomolecules, we direct the reader here https://doi.org/10.1038/s41570-025-00749-9.

How does the covalent deposition of tyramide intermediates enhance spatial localization and signal stability in spectrometry-based imaging workflows?

The covalent deposition ensures that TyrRu is tightly bound to the tyrosine residues surrounding the target biomolecule. Therefore, when the excess solution is washed off, non-specific binding of the complex is reduced. This results in a stable, localized signal that, when analyzed alongside our external standards, is indicative of the concentration of the target biomolecule relative to other samples prepared and analyzed in a similar manner.

What are the major challenges associated with quantifying proteins using LA-ICP-MS imaging, and how do metal-spiked gelatin standards address these challenges?

The standard approach to developing a quantitative analysis is to use matrix-matched calibration standards, and where possible, certified reference materials. This is particularly relevant for LA-ICP-MS, as materials with differing physical characteristics will also exhibit different ablation behavior. To date, there are no suitable biological tissue certified reference materials available for LA-ICP-MS imaging, and calibration standards prepared from homogenized spiked tissues results in standards that are difficult to reproduce and have relatively poor analytical figures of merit. As such, the community has adopted gelatin as a suitable pseudo-matrix. Gelatin is easier to prepare, is homogenous, contains lower background concentrations of the endogenous metals, and has a similar ablation behavior to biological tissue. Some lasers do require the addition of ultraviolet (UV)-absorbent chemical additives to match the ablation behavior, but this addition is quite straightforward during preparation of the standards.

More information on gelatin standards can be found here https://doi.org/10.1016/j.trac.2024.117574 and here https://doi.org/10.1016/j.talanta.2024.127150.

Why might ruthenium tris(bipyridine) complexes be advantageous as multimodal imaging probes for both fluorescence microscopy and mass spectrometry detection?

Ruthenium complexes are excellent for multimodal imaging as ruthenium is exogenous, and therefore have a very low background, they are luminescent, and they are more photostable compared to many standard fluorophores.

How does signal amplification through avidin–biotin–HRP complexes affect sensitivity and signal-to-noise ratio in elemental MSI experiments?

The protocol we developed here combines two separate signal amplification steps, tyramide signal amplification, and avidin-biotin amplification. ICP-MS is a mass-sensitive detector, i.e. the more mass is introduced, the greater the signal. There is a non-linear relationship between the laser shutter size and the mass of sample ablated. That is, with the 20 µm thick sections we used, a 2x2 µm square laser shutter size produces an ablation mass of 80 µm³, compared to 20 µm³ with a 1x1 µm square. TyrRu only contains a single ruthenium atom, in comparison to the commercial Maxpar reagent that contains 30 atoms, therefore in that very small volume of ablated material, the detection capacity of the instrument is low. Both signal amplification methods were necessary to obtain sufficient signal-to-noise that allowed us to image cells in tissue at an incredible 250 nm resolution.

What are the limitations of high-resolution elemental MSI, and how can multimodal fluorescent–metal tags help mitigate these issues?

The major limiting factor of high resolution elemental MSI techniques is the relatively slow speed of analysis when compared to microscopy. Metal tags such as TyrRu allow us to rapidly obtain fluorescent images by microscopy, which is especially useful for optimizing the experimental labelling conditions and to identify regions of interest for quantitative elemental MSI of the target biomolecules.

In multiplexed elemental MSI experiments, how could isotopically enriched metal tags improve analyte discrimination and quantitative accuracy?

Isotopically enriched metal tags are advantageous as they allow us to increase the number of isotopes we can use for highly multiplexed analyses. This is particularly evident during cell phenotyping by imaging mass cytometry, where it is common to see 35-40 biomolecules imaged in a single experiment using enriched lanthanide isotopes as the analytes. The use of isotopically enriched isotopes also increase the detection sensitivity of the analysis.

How might combining immunofluorescence prescreening with LA-ICP-MS imaging optimize experimental throughput and data quality in tissue proteomics studies?

LA-ICP-MS imaging is slower than microscopy. Prescreening via fluorescence microscopy allows us to rapidly identify regions of interest, therefore we can increase our throughput by only imaging these smaller regions by LA-ICP-MS.

References

1. Bergin, R. J.;Kohilas, I.; Lockwood, T. E. et al. TyrRu: A Tyramide-Amplified Multimodal Ruthenium Tag for Elemental Mass Spectrometry and Immunofluorescence Imaging. Chem. Biomed. Imaging 2026. DOI: 10.1021/cbmi.6c00007