PROJECT SUMMARY/ABSTRACT The Sidney Kimmel Comprehensive Cancer Center (SKCCC) Mass Spectrometry Molecular Imaging and Multi-Omics (MSMIMO) Shared Resource (SR) integrates three cooperative facilities across Johns Hopkins: the Applied Imaging Mass Spectrometry (AIMS) facility, the Mass Spectrometry and Proteomics (MSP) facility, and the Metabolomics unit. The MSMIMO SR offers innovative, integrated multiomic workflows that combine omic technologies for systems biology analysis of cancer cells and tissues using state-of-the-art and novel mass spectrometry-based technologies for molecular tissue imaging (spatialomic), metabolomic and proteomic strategies to accelerate cancer research. AIMS offers rapid matrix-assisted laser desorption/ionization (MALDI) imaging of a variety of biomolecules, including endogenous peptides, proteins, N-glycans, lipids, metabolites, and drugs or drug metabolites in cancer cells, spheroids, organoids or tissue sections at high spatial resolution (5 micron). The MSP Facility offers proteomic labeling and label-free strategies, including multidimensional protein identification technology, parallel reaction monitoring, tandem mass tags and stable isotopic labeled amino acids in cell culture for identification and relative or targeted quantification of novel therapeutic proteins, modifications or pathways, dysregulated in cancers, and for tracking interactions of these targets during changes in signal transduction or formation of protein complexes. The Metabolomics Unit offers high throughput detection and quantification of polar and nonpolar metabolites in tissue or fluid extracts. MSMIMO SR services include experimental design, tissue sectioning, sample preparation, buffer exchange, proteolysis, labeling, derivatizations, chromatography, MALDI imaging, tandem mass spectrometry imaging, image analysis, two-dimensional nanoflow liquid chromatography tandem mass analysis, data analysis and interpretation, and assistance with reporting results, training and data submission to public repositories. Biostatisticians and Bioinformaticians are available for in-depth statistical analyses of spatialomic, metabolomic and proteomic results. The MSMIMO SR provides access and training to proprietary software and equipment for SKCCC investigators who wish to perform their own analysis and offers workshops on operating user- accessible mass spectrometers several times a year. The MSMIMO SR's innovative approaches and workflows provide SKCCC Members with the spatial resolution to compare metabolomes, drug metabolism, lipidomes and proteomes across individual cells or cell clusters within the tumor microenvironment. Our comprehensive, highly multiplexed molecular discovery platforms are designed to advance Center Members' research by revealing molecular networks that drive tumorigenesis. During this funding period, 63 SKCCC investigators have used the MSMIMO SR, and more than 30 manuscripts are attributed to this SR. SKCCC Managed Shared R...