The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Cleavable cross-linkers are crucial for chemical cross-linking/mass spectrometry (MS) in protein structural studies.
• Automated data analysis in MS requires reliable cross-linking reagents.

Purpose of the Study:

• Introduce 1,3-diallylurea (DAU) as a novel CID-MS/MS-cleavable, photo-thiol-reactive cross-linker.
• Demonstrate DAU's utility in protein structural analysis.

Main Methods:

• DAU utilizes anti-Markovnikov hydrothiolation with cysteine residues via radical initiation and UV-A irradiation.
• Cross-linking occurs via an orthogonal "click reaction" forming stable alkyl sulfide products.
• Reactions proceed at physiological pH and low temperatures (4 °C).

Main Results:

• DAU is a commercially available, inexpensive reagent.
• The central urea bond in DAU is efficiently cleaved by CID-MS/MS, yielding characteristic product ions.
• Successful cross-linking was demonstrated for peptides, proteins (bMunc13-2, retinal guanylyl cyclase-activating protein-2), and small thiol compounds (cysteine, glutathione).

Conclusions:

• DAU offers a reliable and efficient method for protein structural studies using MS.
• The cleavable nature of DAU enhances automated cross-link identification in MS data analysis.
• DAU represents a significant advancement in cross-linking reagents for structural proteomics.