Crosslinker Nanocarriers-based Intratumoral Delivery for Protein Complex Mapping in Mitochondria of Live Tumor-Bearing Mice

Abstract

Mitochondria, the powerhouses of the cell, are central to tumor initiation and growth, yet their protein interactions in living tumors remain poorly understood. Although crosslinking mass spectrometry (XL-MS) has revealed protein conformations and interactions in cultured cells and isolated tissues, its in vivo application remains limited by poor crosslinker delivery. Here, we present an approach that uses intratumoral injection of crosslinker-loaded nanoparticles in xenografted mice to enable in vivo DSS crosslinking of tumor mitochondria, thereby facilitating XL-MS–based analysis of mitochondrial protein complexes. We identified 2,368 crosslinked peptides from 654 mitochondrial proteins, covering 58% of the human mitochondrial proteome. Among these, 549 crosslinked peptides were mapped to proteins involved in thermogenesis, ribosome function, and branched-chain amino acid degradation—metabolic pathways closely linked to tumor progression. Structural concordance was high (92%) with PDB and AlphaFold models, and complementary conformational insights were obtained, including previously missing data on the DAPIT subunit of ATP synthase, thus complementing cryo-electron microscopy (cryo-EM) structures. We further mapped 217 mitochondrial PPIs, including 146 not annotated in STRING, highlighting the influence of the tumor microenvironment on protein interactions in vivo and providing valuable insights into tumor biology.