MS-Cleavable Cross-Linkers for Protein Complex Mapping
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Solution Overview
Problem
Current methods for structural analysis of protein complexes using cross-linking coupled with mass spectrometry face challenges in unambiguously identifying cross-linked peptides and determining cross-linked sites, due to technical difficulties and limited applicability of existing MS-cleavable cross-linkers.
Innovation Solution
Development of MS-cleavable cross-linkers, such as DSSO, azide-A-DSBSO, and alkyne-A-DSBSO, which include amine-reactive NHS ester groups, CID cleavable bonds, and functional groups for enrichment and click reactions, enabling the mapping of intra- and inter-protein interactions within protein complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing MS-cleavable cross-linkers are used, then cross-linking of protein complexes can be achieved, but unambiguous identification of cross-linked peptides and determination of cross-linked sites remains challenging
Solution Approach 1:
The cross-linker molecule is divided into distinct functional segments: amine-reactive NHS ester groups for protein attachment, a central sulfoxide bond for controlled cleavage, and enrichment handles for affinity purification. This segmentation allows each component to perform its specific function independently, improving identification accuracy while managing complexity through modular design
Solution Approach 2:
The invention changes the chemical parameters of the cross-linker by using specific sulfoxide bonds that exhibit predictable fragmentation behavior under CID conditions. This parameter change ensures that the cross-linker cleaves at defined sites to produce characteristic fragment ions, enabling unambiguous identification of cross-linked peptides and their modification sites through mass spectrometry
2Adaptability or versatility
If multiple types of MS-cleavable cross-linkers are available, then various cross-linking scenarios can be addressed, but complicated synthesis and fragmentation patterns impede wide adaptation
Solution Approach 1:
The cross-linker design incorporates universal amine-reactive NHS ester groups that can attach to lysine residues on any protein, making the reagent broadly applicable to different protein complexes. The enrichment handles provide additional universal functionality for affinity purification, allowing the same cross-linker to be used across diverse applications without requiring custom synthesis for each target
Solution Approach 2:
The invention extracts the complicating factors from the cross-linker design by separating the cross-linking function (NHS ester groups) from the identification function (enrichment handles and controlled fragmentation). This extraction allows the core cross-linking mechanism to remain simple while adding optional enrichment capabilities, thereby improving ease of manufacture and wide adaptation without sacrificing versatility
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These cross-linkers facilitate the identification of cross-linked peptides through tandem mass spectrometry, overcoming previous limitations and enabling detailed structural analysis of protein complexes.
Implementation Method 1
the amine-reactive NHS ester group is designed to react with a lysine side chain in a peptide or a protein
Implementation Method 2
the at least one CID cleavable bond is a sulfoxide bond
Data Source
AI summary
Provided herein is synthesis of novel mass spectrometry-cleavable cross-linking agents. The novel mass spectrometry-cleavable cross-linking agents can be used in mass spectrometry, tandem mass spectrometry, and multi-stage tandem mass spectrometry to facilitate structural analysis of intra-protein interactions in proteins and inter-protein interactions in protein complexes. Also provided are methods of mapping intra-protein interactions in proteins and inter-protein interactions in protein complexes.