Light-Induced Orthogonal Fragmentation of Crosslinked Peptides

Lars Kolbowski, Adam Belsom, Ana M. Pérez-López, Tony Ly, Juri Rappsilber (Lead / Corresponding author)

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Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. 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.

Original languageEnglish
Pages (from-to)2123–2130
Number of pages8
JournalJACS Au
Issue number8
Early online date17 Aug 2023
Publication statusPublished - 28 Aug 2023


  • crosslinkingmass spectrometry
  • ultravioletphotodissociation
  • peptidefragmentation
  • MS3-triggering
  • orthogonalcleavage


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