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2) Twisting Urea- to Imide-Based Mass Spectrometry-Cleavable Cross-Linkers to Embed Affinity Tags

ABSTRACT: Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has been applied to multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas-phase. In this study, we further advance the structural capabilities of DSBU by twisting the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS-cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl dibutyric imide (DSBI) as prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography (IMAC) enrichment. We detail DSBI synthesis and describe its behavior in solution and in the gas-phase while cross-linking isolated proteins and human cell lysates. DSBI and DSBU cross-links are compared at the same enrichment depths to bridge these two cross-linker classes. We validate DSBI cross-links by mapping them in high-resolution structures of large protein assemblies . The cross-links observed yield insights into the morphology of intrinsically disordered proteins (IDPs) and their complexes. The DSBI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christian Ihling

LAB HEAD: Claudio Iacobucci

PROVIDER: PXD050960 | Pride | 2024-09-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DSBI_HEK.fasta	Fasta
	DSBI_HEK293T_Rep1_SEC1_A12.d.zip	Other
	DSBI_HEK293T_Rep1_SEC1_A12.mgf	Mgf
	DSBI_HEK293T_Rep1_SEC1_A12.zhrm	Other
	DSBI_HEK293T_Rep1_SEC1_B1.d.zip	Other

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2) Twisting Urea- to Imide-Based Mass Spectrometry-Cleavable Cross-Linkers to Embed Affinity Tags

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