Significance: Mass spectrometry imaging provides untargeted spatiomolecular information necessary to uncover molecular intratumor heterogeneity. The challenge has been to identify those tumor subpopulations that drive patient outcomes within the highly complex datasets (hyperdimensional data, intratumor heterogeneity, and patient variation). Here we report an automatic, unbiased pipeline to nonlinearly map the hyperdimensional data into a 3D space, and identify molecularly distinct, clinically relevant tumor subpopulations. We demonstrate this pipeline’s ability to uncover subpopulations statistically associated with patient survival in primary tumors of gastric cancer and with metastasis in primary tumors of breast cancer.

MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm² regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.