biostudies-literatureClaes BSRU.S. Department of Health and Human ServicesDutch Province of Limburg2329-2338https://www.ebi.ac.uk/biostudies/studies/S-EPMC9893213biostudies-literatureUnknown95(4)Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavabAnalytical chemistryMALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined.PMC9893213R01 CA213492CA236097Krestensen KKKuik CClaes BSRHeeren RMALim MJVandenbosch MGrgic ARothschild KJYagnik GfalseMALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined.Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavab2023-01-01T00:00:00Z2023 Jan2025-04-26T18:10:39.462Z2025-04-06T15:43:12.582ZS-EPMC98932133663820810.1021/acs.analchem.2c04220