Sequential MALDI-HiPLEX-IHC and Untargeted Spatial Proteomics Mass Spectrometry Imaging to Detect Proteomic Alterations Associated with Tumour Infiltrating Lymphocytes.

MALDI-HiPLEX-IHC mass spectrometry imaging (MSI) represents a newly established workflow to map tens of antibodies linked to photocleavable mass tags (PC-MTs), which report the distribution of antigens in formalin-fixed paraffin-embedded (FFPE) tissue sections. While this highly multiplexed approach has previously been integrated with untargeted methods, the possibility of mapping target cell antigens and performing bottom-up spatial proteomics on the same tissue section has yet to be explored. This proof-of-concept study presents a novel workflow combining MALDI-HiPLEX-IHC with untargeted spatial proteomics to analyze a single FFPE tissue section, using clinical clear cell renal cell carcinoma (ccRCC) tissue as a model. Workflow implementation highlighted the need for an additional antigen retrieval step following antibody staining to aid antibody detachment and enhance tryptic digestion. Moreover, this approach enabled the stratification of histologically similar tumor cores of the same grade based on varying lymphocyte populations, particularly T regulatory cells. Finally, integration with untargeted spatial proteomics revealed proteomic alterations associated with these lymphocyte infiltration patterns. These findings demonstrate the potential of this workflow to map and characterize the molecular environment of tumor-infiltrating lymphocytes, offering insights into the molecular impact of immune cells within the tumor microenvironment.