XLink

XLink analyzes proteolytic products from chemically cross-linked proteins using LC-MALDI tandem mass spectrometry and computational analysis to identify cross-links and derive distance constraints for structural interpretation.

Key Features:

• Cross-Linking Methodology: Employs isotopically coded cross-linkers, including nonlabeled and deuterium-labeled bis-NHS ester reagents, for precise identification of protein-protein contact sites.
• Mass Spectrometry Integration: Optimized for rapid analysis using LC-MALDI tandem mass spectrometry on low microgram amounts of proteins and multimeric complexes and distinguishes Type 0 (monolinks), Type 1, and Type 2 (looplinks or cross-links) modified peptides.
• Distance Constraints Analysis: Provides distance constraints to aid calculation of three-dimensional configurations, identification of binding partners, and localization of contact sites between proteins.
• Versatile Application: Applied to the Colicin E7 DNAse/Im7 heterodimeric complex (23 cross-links identified, including six intersubunit links corresponding to spatial proximity in X-ray structures) and to single subunit proteins such as beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A.

Scientific Applications:

• Structural Biology: Provides distance constraints for constructing and validating three-dimensional models of protein complexes.
• Protein-Protein Interaction Studies: Identifies and characterizes cross-link sites to map interaction networks and localize interfaces.
• Drug Discovery and Development: Supplies molecular-level interaction data that can inform design of therapeutic agents targeting specific protein complexes or interfaces.

Methodology:

Combines LC-MALDI tandem mass spectrometry data with computational analysis to identify and classify Type 0/1/2 modified peptides from isotopically coded bis-NHS ester cross-linkers and to derive distance constraints for calculating three-dimensional configurations and localizing intersubunit contact sites.