Mapping spatial proximities of sulfhydryl groups in proteins using a fluorogenic cross-linker and mass spectrometry

Andrea Sinz, Kuan Wang

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Chemical cross-linking of proteins in combination with mass spectrometric analysis of the reaction products has gained renewed interest as a method of obtaining distance constraints within a protein and determining a low-resolution three-dimensional structure. We present a method for identifying spatially close sulfhydryl groups in proteins employing chemical cross-linking with the fluorogenic, homobifunctional cross-linker dibromobimane, which cross-links thiol pairs within ∼3-6Å. The applicability of our strategy was demonstrated by cross-linking the sulfhydryl groups of Cys-18 and Cys-78 in γ-crystallin F, which are within a distance of 3.57Å according to the X-ray structure. Intramolecularly cross-linked γ-crystallin was first separated from reaction side products by reversed-phase chromatography on a C-4 column. Subsequently, the fraction containing the reacted protein was enzymatically digested with trypsin, and the resulting peptide mixture was separated by a second reversed-phase chromatographic step on a C-18 column, in which the cross-linked peptides were tracked by their fluorescence. The cross-linking product between Cys-18 and Cys-78 in γ-crystallin F was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This strategy presents a rapid method for mapping sulfhydryl groups separated by a distance of ∼3-6Å within a protein.

Original languageEnglish
Pages (from-to)27-32
Number of pages6
JournalAnalytical Biochemistry
Volume331
Issue number1
DOIs
Publication statusPublished - Aug 1 2004
Externally publishedYes

Keywords

  • Chemical cross-linking
  • Dibromobimane
  • Fluorescence
  • HPLC
  • MALDI-TOF mass spectrometry
  • γ-Crystallin

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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