Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry

Proteomics. 2011 Oct;11(20):4109-15. doi: 10.1002/pmic.201100015. Epub 2011 Sep 6.


MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14 Å was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19 Å of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Cross-Linking Reagents / chemistry*
  • Crystallography, X-Ray
  • Dimerization
  • Models, Biological
  • Molecular Sequence Data
  • Molecular Structure
  • Peptides / chemistry*
  • Protein Binding
  • Pseudomonas aeruginosa / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*


  • Bacterial Proteins
  • Cross-Linking Reagents
  • Peptides