ETD-Cleavable Linker for Confident Cross-linked Peptide Identifications

Area of Science:

• Mass Spectrometry
• Proteomics
• Chemical Biology

Background:

• Identifying peptide cross-links is crucial for understanding protein complex structures.
• Existing methods for cross-link identification can be challenging, especially for distinguishing from dead-end products.

Purpose of the Study:

• To demonstrate that diethyl suberthioimidate (DEST) cross-links are cleavable by electron-transfer dissociation (ETD).
• To establish a reliable method for identifying DEST cross-links in protein complexes using ETD mass spectrometry.

Main Methods:

• Formation of peptide cross-links using the homobifunctional linker diethyl suberthioimidate (DEST).
• Analysis of DEST cross-linked peptides using ETD MS2 and MS3 mass spectrometry.
• Characterization of specific mass pairs (peptide-NH2• and peptide+linker+NH3) and reporter ions.

Main Results:

• DEST cross-links are cleavable under ETD conditions.
• ETD MS2 spectra show characteristic mass pairs, aiding cross-link identification.
• Backbone cleavages are more evenly distributed across peptides in DEST cross-links compared to collisional activation.
• Intense reporter ions effectively diagnose dead-end products, distinguishing them from true cross-links.
• ETD mass pairs in MS3 experiments confirm cross-link identifications.

Conclusions:

• ETD mass spectrometry provides a simple and reliable method for identifying DEST cross-links.
• This approach facilitates the study of protein complex architecture and interactions.