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Evaluating Imide-Based Mass Spectrometry-Cleavable Cross-Linkers for Structural Proteomics Studies.

Alessio Di Ianni^1,2, Christian H Ihling^1,2, Tomáš Vranka³

¹Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale D-01620, Germany.

|August 30, 2024

Summary

Disuccinimidyl disuccinic imide (DSSI) is a novel mass spectrometry-cleavable cross-linker. It enables affinity purification and structural biology insights, advancing protein complex analysis.

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Area of Science:

Biochemistry
Structural Biology
Chemical Biology

Background:

Disuccinimidyl dibutyric urea (DSBU) is an MS-cleavable cross-linker used in structural biology and interactomics.
DSBU's urea group facilitates rapid cross-link identification via gas-phase dissociation.

Purpose of the Study:

To introduce a novel class of MS-cleavable cross-linkers based on an imide modification of DSBU.
To present disuccinimidyl disuccinic imide (DSSI) as a prototype, featuring an affinity purification handle.
To evaluate DSSI's utility in structural biology applications, including protein complex analysis.

Main Methods:

Synthesis and characterization of DSSI.
Assessment of DSSI's behavior in solution and gas phase.
Cross-linking of isolated proteins and human cell lysates.
Comparison of DSSI and DSBU cross-linking efficiency.
Validation of DSSI cross-links in high-resolution structures.

Main Results:

DSSI is successfully synthesized and demonstrates MS-cleavability via its imide function.
The phosphonate handle on DSSI enables effective immobilized metal ion affinity chromatography (IMAC) enrichment.
DSSI cross-links provide structural insights into protein assemblies and intrinsically disordered proteins.
DSSI offers comparable or improved performance to DSBU in certain applications.

Conclusions:

DSSI represents a novel class of MS-cleavable and enrichable cross-linkers.
DSSI expands the toolkit for structural biology and interactomics studies.
This new class of cross-linkers holds promise for advancing the understanding of protein structures and interactions.