(M1330-03-16) Benchtop MALDI Imaging of Human Tonsil Proteins Using IHC Probes

Purpose: In the process of drug target discovery, the simple presence of aberrant proteins in patient samples can be an important biomarker, while the spatial distribution of these proteins within tissue may be key to understanding disease progression and providing targeted therapies.  Here, we demonstrate a MALDI imaging solution to allow the precise mapping of proteins using MALDI-IHC imaging probes. Matrix assisted laser desorption ionization immunohistochemistry (MALDI-IHC) is a technique which uses a photocleavable peptide mass tag conjugated with an antibody to label proteins within a tissue sample. Subsequent cleavage of the mass tag allows for this to be detected by MALDI-TOF. Using mass spectrometry imaging (MSI), spatial information of the cleaved peptide can be determined and, from this, the locations of the antibodies, and hence the target proteins in the tissue, can be inferred. Through MALDI-IHC, we can further our knowledge of cellular interactions which is critical to the development new treatments and the Shimadzu benchtop MALDI instruments are ideal platforms to begin exploring the world of MALDI-IHC. In this study, we have analyzed FFPE human tonsil sections using both a 6-plex and 20-plex antibody probe panels at a lateral resolution of 30 μm, demonstrating a reliable low-cost approach with good quality results.

Methods: FFPE human tonsil samples (AMSBIO, Oxford, UK) were mounted on poly-Lysine coated FlexiVision-mini ITO slides. Initially, the protocol was validated using a 6-plex antibody probe mix. To prepare the tissue for analysis, the samples were deparaffinized using xylene. The slides were then immersed in a series of wash solutions (Ethanol: aqueous in varying concentrations) prior to antigen retrieval through exposure to an alkaline buffer. The samples were then stained with a photocleavable mass tag (PC-MT) labelled antibody probe mixture using the workflow provided with the Miralys MALDI HiPLEX-IHC kit (Ambergen, MA, USA). Tissue samples were exposed to UV-light to cleave the mass tags before coating with 2,5 dihydroxybenzoic acid (DHB) using the iMLayer matrix vapor deposition system (Shimadzu Corporation) and recrystallized prior to analysis. Imaging analysis was performed on a benchtop MALDI-TOF instrument (MALDI-8020, Shimadzu Corporation) (Fig. 1). 6-plex acquisition parameters are shown in table format. There were a total of 60201 profiles which were acquired over a period of 4 hours 8 minutes.  20-plex acquisition parameters are shown in table format. There were a total of 27300 profiles which were acquired over a period of 1 hour 52 minutes.  Imaging data was processed using IonView and IMAGEREVEAL MS software packages (Shimadzu Corporation).

Results: Following probe application and cleavage, samples were coated with DHB matrix using varying sublimation times. Alternate timings and temperatures for recrystallisation were also investigated. These parameters were found to have a significant impact on sensitivity and resolution. Optimal results were achieved with a matrix sublimation time of 4 minutes (an approximate coating of 0.22 mg/cm²) and recrystallisation at 3 minutes(5% IPA, 55 ˚C).  Isotopic resolution was easily achieved with a low level of background noise. Follicular hyperplasia is a benign condition which is characterized by an increased number of germinal centers with variably sized mantle zones. Ki67 is recognized as a useful diagnostic marker in immunohistochemistry for follicular hyperplasia, allowing identification and mapping of cell proliferation.  Representative overlaid images are shown demonstrating the different patterns of expression seen in key markers of cell differentiation and T cell activity between normal tissue and tissue with follicular hyperplasia. After imaging analysis, the matrix was removed using acetone and the tissue sections underwent H&E staining. Comparison of the H&E stained images with the ion overlays of the 6-plex probe mixture showed corresponding structures which aligned closely when overlaid.

Conclusion: We have successfully mapped multiplexes of both 6- and 20-peptide mass tags by MALDI-MSI. We have also demonstrated mass spectrometry imaging of follicular hyperplasia of the tonsil which showed different patterns of cell distribution in a key proliferation protein marker. MALDI-MSI is a versatile technique capable of revealing valuable information on biochemically significant compound distributions within a wide variety of samples. In combination with MALDI-IHC probes, the ease with which a range of molecules can be imaged has been extended significantly. Requiring a minimal sample size, MALDI-MSI is proving itself to be a valuable tool for research, facilitating a greater understanding of cellular interactions which is critical to the development new treatments.