Multidimensional Cross-Linking and Real-Time Informatics for Multiprotein Interaction Studies.
Jared P MohrArianne CaudalRong TianJames E BrucePublished in: Journal of proteome research (2023)
Chemical cross-linking combined with mass spectrometry is a technique used to study protein structures and identify protein complexes. Traditionally, chemical cross-linkers contain two reactive groups, allowing them to covalently bond a pair of proximal residues, either within a protein or between two proteins. The output of a cross-linking experiment is a list of interacting site pairs that provide structural constraints for modeling of new structures and complexes. Due to the binary reactive nature of cross-linking reagents, only pairs of interacting sites can be directly observed, and assembly of higher-order structures typically requires prior knowledge of complex composition or iterative docking to produce a putative model. Here, we describe a new tetrameric cross-linker bearing four amine-reactive groups, allowing it to covalently link up to four proteins simultaneously and a real-time instrument method to facilitate the identification of these tetrameric cross-links. We applied this new cross-linker to isolated mitochondria and identified a number of higher-order cross-links in various OXPHOS complexes and ATP synthase, demonstrating its utility in characterizing complex interfaces. We also show that higher-order cross-links can be used to effectively filter models of large protein assemblies generated by using Alphafold. Higher-dimensional cross-linking provides a new avenue for characterizing multiple protein interfaces, even in complex samples such as intact mitochondria.
Keyphrases
- protein protein
- mass spectrometry
- high resolution
- amino acid
- binding protein
- small molecule
- magnetic resonance imaging
- magnetic resonance
- deep learning
- computed tomography
- ionic liquid
- reactive oxygen species
- high performance liquid chromatography
- gas chromatography
- liquid chromatography
- transition metal
- solid phase extraction