Light-Induced Orthogonal Fragmentation of Crosslinked Peptides.

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Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.", "claims": [{"id": "claim-method", "statement": "Kernel density estimate distribution of the peptide backbone fragment intensity vs the cleaved crosslinker peptide stub intensity for the (a) UCCL and (b) DSSO datasets.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:37654600", "locator": "sections[4] \"Mass Spectrometric Fragmentation of UCCL-Linked Peptides\" paragraph 3", "quote": "Kernel density estimate distribution of the peptide backbone fragment intensity vs the cleaved crosslinker peptide stub intensity for the (a) UCCL and (b) DSSO datasets."}], "state": "complete"}, {"id": "claim-data-access", "statement": "All proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE27 partner repository with the dataset identifiers PXD040267 and 10.6019/PXD040267", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:37654600", "locator": "sections[11] \"Supporting Information Available\" paragraph 3", "quote": "All proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE27 partner repository with the dataset identifiers PXD040267 and 10.6019/PXD040267"}], "state": "complete"}, {"id": "claim-target-result", "statement": "The UVPD-cleavable UCCL crosslinker increased the crosslinker-to-peptide fragment intensity ratio to 5 and improved the selection of individual peptides for MS3 fragmentation.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:37654600", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "The UVPD-cleavable UCCL crosslinker increased the crosslinker-to-peptide fragment intensity ratio to 5 and improved the selection of individual peptides for MS3 fragmentation.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:37654600"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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