Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions

Chang Ho Sohn, Heather D Agnew, J Eugene Lee, Michael J Sweredoski, Robert L J Graham, Geoffrey T Smith, Sonja Hess, Gregg Czerwieniec, Joseph A Loo, James R Heath, Raymond J Deshaies, J L Beauchamp

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)


We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability.

Original languageEnglish
Pages (from-to)2662-9
Number of pages8
JournalAnalytical Chemistry
Issue number6
Publication statusPublished - 20 Mar 2012


  • Amino Acid Sequence
  • Avidin/chemistry
  • Chromatography, Affinity
  • Cross-Linking Reagents/chemistry
  • HEK293 Cells
  • Humans
  • Mass Spectrometry/methods
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides/chemistry
  • Proteins/chemistry
  • Proteomics/methods
  • Ubiquitin/chemistry


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