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

Andrea Sinz, Kuan Wang

研究成果: 雜誌貢獻文章同行評審

24 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)27-32
頁數6
期刊Analytical Biochemistry
331
發行號1
DOIs
出版狀態已發佈 - 8月 1 2004
對外發佈

ASJC Scopus subject areas

  • 生物物理學
  • 生物化學
  • 分子生物學
  • 細胞生物學

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