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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.

Trapped ion mobility spectrometry (TIMS) using parallel accumulation serial fragmentation (PASEF ® ) is an advanced analytical technique that offers several advantages in mass spectrometry (MS)-based lipidomics. TIMS provides an additional dimension of separation to mass spectrometry and accurate collision cross-section (CCS) measurements for ions, aiding in the structural characterization of molecules. This is especially valuable in lipidomics for identifying and distinguishing isomeric or structurally similar compounds. On the other hand, PASEF technology allows for fast and efficient data acquisition by accumulating ions in parallel and then serially fragmenting them. This accelerates the analysis process and improves throughput, making it suitable for high-throughput applications. Moreover, the combination of TIMS and PASEF reduces co-elution and ion coalescence issues, leading to cleaner and more interpretable mass spectra. This results in higher data quality and more confident identifications. In this chapter, a data-dependent TIMS-PASEF ® workflow for lipidomics analysis is presented., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)