Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes

Mol Cell Proteomics. 2005 Aug;4(8):1167-79. doi: 10.1074/mcp.T400016-MCP200. Epub 2005 May 18.

Abstract

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Algorithms
  • Cross-Linking Reagents / chemical synthesis
  • Cross-Linking Reagents / chemistry*
  • Deuterium / chemistry
  • HIV Reverse Transcriptase / analysis*
  • Humans
  • Peptide Fragments / chemistry*
  • Protein Binding
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Succinimides / chemical synthesis
  • Succinimides / chemistry*

Substances

  • Cross-Linking Reagents
  • Peptide Fragments
  • Succinimides
  • ethylene glycolylbis(succinimidyl succinate)
  • Deuterium
  • HIV Reverse Transcriptase