Developing new isotope-coded mass spectrometry-cleavable cross-linkers for elucidating protein structures.

Area of Science:

• Structural Biology
• Biochemistry
• Analytical Chemistry

Background:

• Understanding protein complex structure is crucial for function and regulation.
• Existing tools for structural characterization have limitations.
• Cross-linking mass spectrometry (XL-MS) is a powerful technique for studying protein interactions and complex topology.

Purpose of the Study:

• To develop novel isotope-coded, MS-cleavable homobifunctional cross-linkers.
• To enhance the capabilities of XL-MS for structural analysis of protein complexes.
• To establish a new XL-MS workflow for studying protein complex dynamics.

Main Methods:

• Development and synthesis of d0- and d10-labeled dimethyl disuccinimidyl sulfoxide (DMDSSO) cross-linkers.
• Characterization of DMDSSO cross-linked peptides using mass spectrometry (MS2 and MS3 analysis).
• Concurrent usage of isotope-coded cross-linkers for quantitative analysis.

Main Results:

• Sulfoxide-containing MS-cleavable cross-linkers provide robust and predictable MS2 fragmentation.
• MS3 analysis simplifies and enables unambiguous identification of cross-linked peptides.
• Concurrent use of d0/d10-DMDSSO yields a characteristic doublet pattern, increasing identification confidence.
• The isotopic profile allows for quantitative analysis of cross-linked peptides.

Conclusions:

• The developed DMDSSO cross-linkers represent a significant advancement for XL-MS.
• This new XL-MS workflow facilitates robust identification and quantification of protein interactions.
• The methodology enables the study of both static and dynamic aspects of protein complex structures.