Mass Spectrometry Imaging–Guided Proteomics, an Emerging Frontier in Spatial Biology

ABSTRACT Spatial heterogeneity is a defining feature of tissue organization, yet conventional proteomic analysis provides limited information about the spatial origins of identified proteins. Spatially guided proteomics addresses this limitation by using tissue imaging techniques to select regions of interest (ROIs) for localized analysis by liquid chromatography‐tandem mass spectrometry (LC–MS/MS). Among the imaging modalities available for this purpose, mass spectrometry imaging (MSI) stands out for its ability to map diverse molecular species directly within tissue without predefined targets, allowing chemically distinct regions to guide tissue sampling. By bridging spatial molecular profiles with high protein coverage, MSI‐guided proteomics offers comprehensive biological insight. This review examines the workflow from MSI data acquisition through ROI definition, image registration, tissue isolation, and sample preparation to LC–MS/MS analysis. We compare common MSI modalities, discuss methods for ROI isolation, and summarize advances in nanoproteomics methods that support analysis of tissue samples at the microscopic scale. We critically evaluate applications in cancer research, infectious disease, neuroscience, and environmental science. Finally, we discuss the challenges and future directions of MSI‐guided proteomics. We conclude that ongoing developments in the MSI‐guided proteomics workflow position it as an emerging strategy for investigating localized biological processes within heterogeneous tissues.