Despite a steady stream of technological breakthroughs, the translation of advanced analytical tools into routine pathology practice continues to face significant hurdles. While genomics and digital pathology have made strong inroads, other disciplines such as mass spectrometry (MS) – long recognized for their analytical power – have struggled to gain widespread adoption in the clinical diagnostic space.
This is especially evident in the case of mass spectrometry imaging (MSI), a technique capable of generating detailed molecular maps of tissue surfaces. First introduced in the late 1990s, MSI was heralded as a potential game-changer for pathology. Yet, more than two decades later, it remains largely confined to research.
The reasons for this are not primarily technical limitations, but practical misalignments between MSI workflows and the realities of clinical pathology. Traditional MSI has relied heavily on fresh-frozen or flash-frozen samples, which are rarely used in routine diagnostics. In contrast, most pathology laboratories rely on formalin-fixed paraffin-embedded (FFPE) tissue – the clinical gold standard for decades due to its robustness, ease of storage, and compatibility with histological and immunohistochemical analysis.
Unfortunately, early attempts to adapt MSI to FFPE specimens met with limited success. It was widely believed that the crosslinking effects of formaldehyde fixation rendered many biomolecules inaccessible to MS analysis. Furthermore, standard FFPE processing steps (eg, dehydration, clearing, and embedding) are known to strip out lipid molecules and other (classes of) analytes of interest. As a result, the field moved away from FFPE in favor of frozen tissue, effectively excluding the vast archive of pathology samples from the potential benefits of MSI.
However, recent work has upended this assumption. Our research (1–4) has demonstrated that it is not only possible, but highly effective, to perform high-resolution MSI of especially small and labile compounds on archived FFPE tissues, including blocks stored for over 30 years. Using optimized protocols and cutting-edge instrumentation – such as Orbitrap Fourier-transform mass spectrometers coupled with atmospheric pressure matrix-assisted laser desorption ionization (AP/MALDI) sources – we have successfully imaged a variety of physiologically relevant biomolecules, including endogenous neuropeptides and polar metabolites, directly from FFPE sections. These analytes were recovered in their intact, post-translationally modified forms, defying previous assumptions.
This capability, which we call mass spectrometry histochemistry (MSHC), mirrors the workflow of immunohistochemistry (IHC) while offering vastly improved molecular specificity and spatial resolution (down to 5×5 μm²). Importantly, it can be applied directly to the millions of FFPE tissue blocks stored in biobanks worldwide.
The potential of MSHC is huge; we can now analyze archived samples from biobanks to find small molecule and peptide biomarkers linked to how serious a disease is, how patients respond to treatment, or what their long-term outlook might be. Since MSHC works directly on the same slides already used for routine pathology, it fits easily into existing lab workflows – making it much simpler to bring into clinical practice.
With this in mind, I propose a three-phased rollout of MSHC into pathology:
Retrospective Disease Typing and Biomarker Discovery: Leveraging well-annotated biobank samples, MSHC enables retrospective analysis of diseases that remain difficult to classify histologically. In one ongoing study, we’re exploring how metabolite ion patterns can distinguish between highly aggressive and indolent tumor types – something current IHC markers cannot reliably do. Similarly, this approach also opens the door to studying tissues from patients responsive to a specific treatment versus irresponsive ones and even including diseases only confirmed post mortem, such as certain neurodegenerative disorders.
Validation for Clinical Use: Once candidate molecular signatures are validated, MSHC can operate in parallel with IHC to enhance diagnostic accuracy. Crucially, this does not require altering current specimen workflows.
Predictive and Prognostic Testing: Even without identifying individual molecules, MSHC mass signatures can serve as robust diagnostic tools – just as unidentified protein patterns are used today in MALDI-TOF microbial ID systems. Identified biomarkers may also be translated into minimally invasive assays for serum, plasma, urine, or even tear fluid, further expanding their clinical utility.
The promise of MSHC is no longer theoretical. The tools exist, the protocols are feasible, and the samples are waiting. What’s needed now is collaborative engagement from the pathology community – to help select cases, validate findings, and integrate this new layer of molecular insight into everyday diagnostics.
In short, MSHC represents a rare convergence of scientific innovation and clinical relevance. It transforms the existing pathology archive into a living resource for discovery and diagnosis, offering the potential to revolutionize how we classify disease and tailor treatment.
As pathology moves toward a multi-omics future, MSHC deserves a central role. Now is the time to unlock the diagnostic potential of the archives.
Peter Verhaert is Founder of ProteoFormiX, Part-time Professor of the Department Imaging and Pathology (Translational Cell & Tissue Research Unit) at the University of Leuven Faculty of Medicine, Belgium, and Visiting Professor at the University of Witwatersrand, South Africa.
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References
- MRL Paine et al., Anal Chem, 90, 15 (2018). PMID: 29975508.
- G Frache et al., Methods Mol Biol (2025). PMID: 40498187.
- YL Cintron-Diaz et al., J Am Soc Mass Spectrom, 33, 4 (2022). PMID: 35258288.
- P Verhaert et al., “High Resolution Mass Spectrometry of Cystine-Containing Neuropeptides in Histological Sections of Human FFPE Tissue Banks,” in Cysteine – New Insights. IntechOpen (2024). DOI: 10.5772/intechopen.1004948.
