A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides

Nat Commun. 2019 Jul 30;10(1):3404. doi: 10.1038/s41467-019-11337-z.


We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. With a two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, using simulated datasets, synthetic datasets, 15N metabolically labeled datasets, and entrapment databases, four analysis methods were designed to evaluate the credibility of ten state-of-the-art search engines. This systematic evaluation shows that pLink 2 outperforms these methods in precision and sensitivity, especially at proteome scales. Lastly, re-analysis of four published proteome-scale cross-linking datasets with pLink 2 required only a fraction of the time used by pLink 1, with up to 27% more cross-linked residue pairs identified. pLink 2 is therefore an efficient and reliable tool for cross-linking mass spectrometry analysis, and the systematic evaluation methods described here will be useful for future software development.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • Databases, Protein
  • Humans
  • Peptides / chemistry*
  • Proteome / chemistry*
  • Proteomics
  • Search Engine / methods*
  • Software


  • Peptides
  • Proteome