Understanding the molecular basis of disease pathology or tissue homeostasis relies on knowing how thousands of biomolecules, including proteins, nucleic acids, lipids, and metabolites, interact in cells and tissues. Uncovering the identity and spatial organization of these molecules that are involved in human biology and disease is vital for deepening this knowledge and developing new and effective diagnostics and therapies. The importance of analysing molecules in a spatial context has gained the attention of researchers across the globe.
Now, with spatial information seen as a vital component when describing the microenvironment of disease in tissues and determining the location and interactions of cellular components that dictate disease outcome, a collection of diagnostic tools has become established. This commonly includes immunohistochemistry (IHC), imaging mass cytometry (IMC) and, more recently, spatial transcriptomics. Traditional histopathology methods such as fluorescence based IHC only allow spatial mapping of a few biomarkers at a time, which stems from the intrinsic nature of molecular fluorophores that typically exhibit relatively broad excitation and emission bands, thus preventing the detection of more than a few individual fluorophore labels with high selectivity.
The current scientific tools space does not offer solutions for acquiring spatial distribution and association of both small untargeted molecules and large targeted molecules from single tissue sections. Different sections, even from adjacent tissue slices, do not provide the full picture due to morphology-based challenges in merging multiple images. While imaging small molecules using MALDI Imaging has become routine, it has not been possible to do this routinely with proteins using a simple and rapid workflow.
By combining IHC methods, which can target specific proteins together with MALDI Imaging that offers high multiplexing on a single tissue, a new method, known as MALDI HiPLEX-IHC, is emerging. Although the concept of applying both techniques in series is not new, combining IHC and MALDI Imaging on one tissue to provide multi-omics mapping of small molecules and intact proteins has not been successfully applied until now.
Figure 1: Basic steps used for MALDI HiPLEX-IHC tissue imaging enabling both MALDI-MSI of untargeted small molecules and MALDI-MSI and fluorescence images of targeted intact expressed proteins to be obtained using MiralysTM PC-MT antibody probes all on the same tissue specimen
The three key features of the MALDI HiPLEX technology:
Multiplexing – image more than multiple biomarkers in a single run from a large region of interest up to the size of the microscope slide
Multiomics – measure small molecules, intact proteins, other macromolecules, and PTMs in one workflow
Multimodality – gather information from many imaging modalities, from one sample.
Label-free untargeted small-molecule MALDI Imaging can directly analyze lipids, drugs, and metabolites, which is not possible using standard IHC or IMC. This capability will allow researchers and pathologists to co-localize small-molecule drugs and drug-targets, such as their receptors, as well as associated biomolecules involved in the cellular response to the drug. Metabolic pathways associated with known proteins of interest can be spatially mapped and protein activity can be assessed quantitatively.