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Crosslinker nanocarrier-based intratumoral delivery for protein complex mapping in mitochondria of live tumor-bearing
Yuwan Chen1,2, Wenxin Fu1,3, Baofu Ma1,2
1State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences Dalian 116023 China lihuazhang@dicp.ac.cn yangkaiguang@dicp.ac.cn.
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 2368 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.
Insights
Researchers developed a novel nanoparticle delivery method for in vivo crosslinking mass spectrometry, revealing new mitochondrial protein interactions crucial for tumor growth. This advance enhances our understanding of tumor biology and mitochondrial function in living cancers.
Area of Science:
- Mitochondrial biology
- Proteomics
- Cancer research
Background:
- Mitochondria play a key role in tumor progression, but their protein interactions within living tumors are not well understood.
- In vivo crosslinking mass spectrometry (XL-MS) is limited by challenges in delivering crosslinking agents to tumors.
Purpose of the Study:
- To develop an effective method for in vivo crosslinking of mitochondrial proteins in tumors.
- To analyze mitochondrial protein complexes and interactions within living tumors using XL-MS.
Main Methods:
- Intratumoral injection of crosslinker-loaded nanoparticles into xenografted mouse models.
- In vivo dithiobis(succinimidyl propionate) (DSS) crosslinking of tumor mitochondria.
- XL-MS analysis to identify crosslinked peptides and protein-protein interactions (PPIs).
Main Results:
- Identified 2368 crosslinked peptides from 654 mitochondrial proteins, covering 58% of the human mitochondrial proteome.
- Mapped 549 crosslinked peptides to proteins in thermogenesis, ribosome function, and amino acid degradation pathways linked to tumor progression.
- Discovered 217 mitochondrial PPIs, including 146 novel interactions, providing insights into the tumor microenvironment's influence.
Conclusions:
- The nanoparticle-based approach enables effective in vivo mitochondrial crosslinking and XL-MS analysis in tumors.
- This method reveals novel mitochondrial protein interactions and pathways critical for tumor biology.
- The findings complement existing structural data and offer new avenues for cancer research.
