Formalin-fixed paraffin-embedded (FFPE) tissue represents the primary source of clinical tissue and is routinely used in MALDI-MSI studies. However, it is not particularly suitable for lipidomics imaging given that many species are depleted during tissue processing. Irrespective, a number of solvent-resistant lipids remain, but their extraction may be hindered by the cross-link between proteins. Therefore, an antigen retrieval step could enable the extraction of a greater number of lipids and may provide information that is complementary to that which can be obtained from other biomolecules, such as proteins. In this short communication, we aim to address the effect of performing antigen retrieval prior to MALDI-MSI of lipids in FFPE tissue. As a result, an increased number of lipid signals could be detected and may have derived from lipid species that are known to be implicated in the lipid–protein cross-linking that is formed as a result of formalin fixation. Human renal cancer tissue was used as a proof of concept to determine whether using these detected lipid signals were also able to highlight the histopathological regions that were present. These preliminary findings may highlight the potential to enhance the clinical relevance of the lipidomic information obtained from FFPE tissue.

Purpose Matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) technology has advanced rapidly during recent years with the development of instruments equipped with low‐diameter lasers that are suitable for high spatial resolution imaging. This may provide significant advantages in certain fields of molecular pathology where more specific protein fingerprints of individual cell types are required, such as renal pathology. Experimental design Here MALDI‐MSI analysis of a cohort of membranous nephropathy (MN) patients is performed among which patients either responded favorably (R; n = 6), or unfavorably (NR; n = 4), to immunosuppressive treatment (Ponticelli Regimen), employing a 10 µm laser spot diameter. Results Specific tryptic peptide profiles of the different cellular regions within the glomerulus can be generated, similarly for the epithelial cells belonging to the proximal and distal tubules. Conversely, specific glomerular and sub‐glomerular profiles cannot be obtained while using the pixel size performed in previous studies (50 µm). Furthermore, two proteins are highlighted, sonic hedgehog and α‐smooth muscle actin, whose signal intensity and spatial localization within the sub‐glomerular and tubulointerstitial compartments differ between treatment responders and non‐responders. Conclusions and clinical relevance The present study exemplifies the advantage of using high spatial resolution MALDI‐MSI for the study of MN and highlights that such findings have the potential to provide complementary support in the routine prognostic assessment of MN patients.

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