Optimizing the enrichment of cross-linked products for mass spectrometric protein analysis

Rapid Commun Mass Spectrom. 2012 Mar 30;26(6):653-8. doi: 10.1002/rcm.6150.


Rationale: Chemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography.

Methods: SCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS(3)) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins.

Results: Conditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS(3), 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS(3) allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment.

Conclusions: For both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Chromatography, Ion Exchange / methods*
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / isolation & purification*
  • Escherichia coli / enzymology
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / isolation & purification*
  • Models, Molecular
  • Peptides / chemistry
  • Peptides / isolation & purification*
  • Serum Albumin, Bovine / chemistry
  • Serum Albumin, Bovine / isolation & purification*
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Succinates / chemistry
  • Succinimides / chemistry
  • Tandem Mass Spectrometry / methods


  • Cross-Linking Reagents
  • Peptides
  • Succinates
  • Succinimides
  • Serum Albumin, Bovine
  • Glutathione Transferase
  • N-hydroxysuccinimide