Probability-based shotgun cross-linking sites analysis

Application Note


We recently developed a shotgun tool for cross-linking sites analysis, X!Link, for the sensitive and high-throughput analysis of chemically cross-linked proteins or multiprotein complexes (J. Proteome Res. 2007, 6, 3908–3917). Here, we report a further development of the tool using a probability-based scoring system. It calculates explicit E-values, with which sensitive detection of the cross-links is possible with very low false positives, and now can be applied to moderate numbers of protein sequences. Most of the false positives in large scale analysis originate from partial matching where one side of the peptides is correctly matched while the other side is incorrectly matched. Additional E-values were calculated for each peptide and effectively minimized false positives from such partial matching. The usefulness of the new scoring system was demonstrated for a previously published dataset from a cross-linked cytochrome c protein, searching against a large database of equine protein sequences.

Supplementary material

13361_2011_201001896_MOESM1_ESM.pdf (177 kb)
Supplementary material, approximately 181 KB.


  1. 1.
    Sinz, A. Chemical Cross-Linking and Mass Spectrometry to Map Three-Dimensional Protein Structures and Protein-Protein Interactions. Mass Spectrom. Rev. 2006, 25, 663–682.CrossRefGoogle Scholar
  2. 2.
    Huang, B. X.; Kim, H. Interdomain Conformational Changes in Akt Activation Revealed by Chemical Cross-linking and Tandem Mass Spectrometry. Mol. Cell Proteom. 2006, 5, 1045–1053.CrossRefGoogle Scholar
  3. 3.
    Seebacher, J.; Mallick, P.; Zhang, N.; Eddes, J. S.; Aebersold, R.; Gelb, M. H. Protein Cross-Linking Analysis Using Mass Spectrometry, Isotope-Coded Cross-Linkers, and Integrated Computational Data Processing. J. Proteome Res. 2006, 5, 2270–2282.CrossRefGoogle Scholar
  4. 4.
    Lee, Y. J.; Lachner, L.; Nunnari, J.; Phinney, B. S. Shotgun Cross-Linking Analysis for Studying Quaternary and Tertiary Protein Structures. J. Proteome Res. 2007, 6, 3908–3917.CrossRefGoogle Scholar
  5. 5.
    Eng, J. K.; McCormack, A. L.; Yates, J. R. An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database. J. Am. Soc. Mass Spectrom. 1994, 5, 976–989.CrossRefGoogle Scholar
  6. 6.
    Geer, L. Y.; Markey, S. P.; Kowalak, J. A.; Wagner, L.; Xu, M.; Maynard, D. M.; Yang, X.; Shi, W.; Bryant, S. H. Open Mass Spectrometry Search Algorithm. J. Proteome Res. 2004, 3, 958–964.CrossRefGoogle Scholar
  7. 7.
    Swaim, C. L.; Smith, J. B.; Smith, D. L. Unexpected Products from the Reaction of the Synthetic Cross-Linker 3,3′-Dithiobis(Sulfosuccinimidylpropionate), DTSSP with Peptides. J. Am. Soc. Mass Spectrom. 2004, 15, 736–749.CrossRefGoogle Scholar
  8. 8.
    Rinner, O.; Seebacher, J.; Walzthoeni, T.; Mueller, L.; Beck, M.; Schmidt, A.; Mueller, M.; Aebersold, R. Identification of Cross-Linked Peptides from Large Sequence Databases. Nat. Methods. 2008, 5, 315–318.CrossRefGoogle Scholar
  9. 9.
    Singh, P.; Shaffer, S. A.; Scherl, A.; Holman, C.; Pfuetzner, R. A.; Freeman, T. J. L.; Miller, S. I.; Hernandez, P.; Appel, R. D.; Goodlett, D. R. Characterization of Protein Cross-Links via Mass Spectrometry and an Open-Modification Search Strategy. Anal. Chem. 2008, 80, 8799–8806.CrossRefGoogle Scholar
  10. 10.
    Guo, X.; Bandyopadhyay, P.; Schilling, B.; Young, M. M.; Fujii, N.; Aynechi, T.; Guy, R. K.; Kuntz, I. D.; Gibson, B. W. Partial Acetylation of Lysine Residues Improves Intraprotein Cross-Linking. Anal. Chem. 2008, 80, 951–960.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  1. 1.Ames Laboratory-USDOE and Department of ChemistryIowa State UniversityAmesUSA

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