Brain Landscape In Situ Crosslinking Mass Spectrometry (BLIS-XL-MS) Enables Global Analysis of Protein Structural Remodeling in the Brain

Pathological protein conformational remodeling is a key molecular feature of brain disorders. However, conventional approaches to understand the changes in potein conformation are targeted and difficult to scale proteome-wide analysis. Emerging mass spectrometry-based structural proteomic methods broaden coverage but often involve complex workflows and trade-offs between labeling efficiency and preservation of the in vivo molecular state. Thus, quantitative analysis of proteome-wide structural remodeling remains limited. Here we present Brain Landscape In Situ Crosslinking Mass Spectrometry (BLIS-XL-MS), an experimental and computational framework for analyzing protein and protein-complex remodeling in brain tissue. BLIS-XL-MS stabilizes the molecular state before secondary crosslinking, improves reagent accessibility through sectioning and permeabilization, and integrates quantitative crosslink analysis with structural and protein-interaction-network interpretation. Applied to brains from GluA1A636T knock-in mice, modeling a neurodevelopmental disorder, BLIS-XL-MS revealed coordinated remodeling of protein systems involved in AMPA receptor-trafficking, mitochondrial function and cell-death pathways. We demonstrate that BLIS-XL-MS provides a scalable framework for mapping disease-associated structural remodeling across the brain proteome.