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

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Abstract

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
Volume84
Issue number6
DOIs
Publication statusPublished - 20 Mar 2012

Keywords

  • 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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  • Cite this

    Sohn, C. H., Agnew, H. D., Lee, J. E., Sweredoski, M. J., Graham, R. L. J., Smith, G. T., Hess, S., Czerwieniec, G., Loo, J. A., Heath, J. R., Deshaies, R. J., & Beauchamp, J. L. (2012). Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions. Analytical Chemistry, 84(6), 2662-9. https://doi.org/10.1021/ac202637n