Data-independent acquisition improves quantitative cross-linking mass spectrometry

Fränze Müller, Lars Kolbowski, Oliver M Bernhardt, Lukas Reiter, Juri Rappsilber

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Quantitative cross-linking mass spectrometry (QCLMS) reveals structural detail on altered protein states in solution. On its way to becoming a routine technology, QCLMS could benefit from data-independent acquisition (DIA), which generally enables greater reproducibility than data-dependent acquisition (DDA) and increased throughput over targeted methods. Therefore, here we introduce DIA to QCLMS by extending a widely used DIA software, Spectronaut, to also accommodate cross-link data. A mixture of seven proteins cross-linked with bis[sulfosuccinimidyl] suberate (BS3) was used to evaluate this workflow. Out of the 414 identified unique residue pairs, 292 (70%) were quantifiable across triplicates with a coefficient of variation (CV) of 10%, with manual correction of peak selection and boundaries for PSMs in the lower quartile of individual CV values. This compares favourably to DDA where we quantified cross-links across triplicates with a CV of 66%, for a single protein. We found DIA-QCLMS to be capable of detecting changing abundances of cross-linked peptides in complex mixtures, despite the ratio compression encountered when increasing sample complexity through the addition of E. coli cell lysate as matrix. In conclusion, the DIA software Spectronaut can now be used in cross-linking and DIA is indeed able to improve QCLMS.

Original languageEnglish
Pages (from-to)786-795
Number of pages11
JournalMolecular & Cellular Proteomics (MCP)
Issue number4
Early online date16 Jan 2019
Publication statusE-pub ahead of print - 16 Jan 2019

Keywords / Materials (for Non-textual outputs)

  • Bioinformatics software
  • Data independent acquisition
  • Label-free quantification
  • Mass Spectrometry
  • Protein Cross-linking
  • Quantification
  • Reproducibility
  • Spectronaut
  • Xi


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