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Cleavable Photo-Crosslinker Enables sFFP-Driven Strategy for Mapping HSP90β Oligomer Structure

ABSTRACT: Crosslinking technology is a pivotal tool for resolving dynamic protein interaction networks. While traditional chemical crosslinkers face limitations in sensitivity, reaction kinetics, and in situ activation, cleavable and photo-crosslinkers have emerged as alternatives. However, their complex spectral signatures remain underutilized in database search algorithms. Here, we mechanistically demonstrated the latent cleavable properties of diazirine-based photo-crosslinkers through tandem mass spectrometry. By leveraging side-chain fragmentation fingerprint (sFFP) under a target-decoy strategy, we reduced the false discovery rate (FDR) of BSA crosslinking data from 12% to 2.7%. To synergize cleavable and photo-reactive advantages, we rationally engineered the first dual-cleavable photo-crosslinker BDG, featuring two sFFP signatures (sc/sz and sc'/sz') and rapid in situ photo-activation. Validated by plink3 Score and sFFP analysis, BDG enabled the first photochemical reconstruction of HSP90β oligomer conformations, providing structural insights into chaperone-mediated stress protection. This work establishes a methodological paradigm for probing dynamic protein interactions.

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ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: yida jiang

LAB HEAD: Chun Tang

PROVIDER: PXD064219 | Pride | 2026-04-09

REPOSITORIES: Pride

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Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo-XL-MS has a propensity to capture protein-protein interactions, particularly involving low-abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the “darker” corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.

			Action	DRS
	BSA-BD3_MS2.xlsx	Xlsx
	BSA-BDG_MS2_R1.raw	Raw
	BSA-BDG_MS2_R2.raw	Raw
	BSA-BDG_MS2_R3.raw	Raw
	BSA-BDG_MS3_R1.raw	Raw

Dataset Information

Cleavable Photo-Crosslinker Enables sFFP-Driven Strategy for Mapping HSP90β Oligomer Structure

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