Dataset Information

Reprogramming the Lipid Peroxidation Product 4-ONE as a Chemoselective Cleavable Crosslinker

ABSTRACT: Nature builds and stabilizes proteins through covalent biological glues, yet the logic and traceability of metabolite-driven crosslinking remain obscure. Here we reprogram the lipid peroxidation product 4-oxo-nonenal (4-ONE) into a metabolite-derived Cys-Lys biological glue and mass-cleavable crosslinker. Through a chemoselective two-step pathway, Michael addition to cysteine activates a latent aldehyde that cyclizes with lysine to form a stable pyrrole linkage under physiological conditions. Late-stage oxidation converts this linkage into a mass-cleavable pyrrole sulfoxide, establishing COSMIc (Crosslink Oxidation to Sulfoxide for Mass-Cleavable Interactomics) for site-resolved identification of linked residues through diagnostic sulfenic acid and dehydroalanine fragments. Beyond peptide stapling, macrocyclization, late-stage functionalization, selective protein modification, and illuminating hyperreactive Lys and Cys residues that define 4-ONE reactivity, COSMIc enables detection of structurally informative crosslinks in proteins, proteomes and the human 26S proteasome, where peroxide-induced sulfoxide formation markedly improves fragment assignment and residue-pair confidence. Together, these findings transform 4-ONE from a toxic electrophile into a programmable covalent connector for structural proteomics, covalent mapping and interactome analysis.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Anthony Ciancone

LAB HEAD: Francis J. O'Reilly

PROVIDER: PXD077887 | Pride | 2026-06-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	E260403_02_AMC_RPN1_IP_4ONE_IV_Dose_pepSEC_IDruns_Inj1.raw	Raw
	E260403_03_AMC_RPN1_IP_4ONE_IV_Dose_pepSEC_IDruns_Inj2.raw	Raw
	E260403_04_AMC_RPN1_IP_4ONE_IV_Dose_pepSEC_IDruns_Inj3.raw	Raw
	E260414_01_AMC_RPN1_4ONE_IV_Dose_H2O2_pepSEC_IDs_Inj1.raw	Raw
	E260414_02_AMC_RPN1_4ONE_IV_Dose_H2O2_pepSEC_IDs_Inj2.raw	Raw

Items per page:

1 - 5 of 31

Similar Datasets

Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.

Dataset Information

Reprogramming the Lipid Peroxidation Product 4-ONE as a Chemoselective Cleavable Crosslinker

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets