Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

Area of Science:

• Biochemistry
• Proteomics
• Computational Biology

Background:

• Understanding protein tertiary and quaternary structures is crucial for deciphering biological functions.
• Identifying protein-protein interactions aids in mapping cellular pathways and disease mechanisms.
• Existing methods for analyzing cross-linked proteins can be time-consuming and complex.

Purpose of the Study:

• To develop a novel, sensitive, and high-throughput computational approach for analyzing chemically cross-linked proteins.
• To identify tertiary and quaternary interaction sites in protein complexes using mass spectrometry data.
• To create a faster and simpler method for protein cross-linking analysis compared to current techniques.

Main Methods:

• Direct analysis of digests from chemically cross-linked proteins using high-accuracy LC-MS/MS data.
• Application of a novel computer algorithm, X!Link, to identify cross-links between peptides.
• Utilizing BS3 (bis(sulfosuccinimidyl) suberate) as a chemical cross-linking reagent.

Main Results:

• Identified 31 cross-links (21 interpeptide, 10 intrapeptide) in cytochrome c, detecting all 19 lysine residues.
• Identified 46 cross-links (38 interpeptide, 8 intrapeptide) in the Dnm1G385D homodimer.
• Achieved a low false discovery rate (1%) and demonstrated high sensitivity, detecting 33% of Lys pairs within 20 Å.

Conclusions:

• The developed X!Link algorithm provides a sensitive and rapid MS-based approach for identifying peptide-peptide cross-links.
• This method eliminates the need for isotopic labeling or non-cross-linked controls, offering a simpler workflow.
• Manual inspection is recommended for accurate site assignment, especially with complex protein samples.