Mapping spatial proximities of sulfhydryl groups in proteins using a fluorogenic cross-linker and mass spectrometry.
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 approximately 3-6A. The applicability of our strategy was demonstrated by cross-linking the sulfhydryl groups of Cys-18 and Cys-78 in gamma-crystallin F, which are within a distance of 3.57A according to the X-ray structure. Intramolecularly cross-linked gamma-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 gamma-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 approximately 3-6A within a protein.
Sinz
Andrea
A
Muscle Proteomics and Nanotechnology Section, Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. sinz@chemie.uni-leipzig.de
Wang
Kuan
K
D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D005453:Fluorescence; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D038222:gamma-Crystallins / Q000737:chemistry*
D001643:Bridged Bicyclo Compounds; D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D038222:gamma-Crystallins; C037094:dibromobimane; D003545:Cysteine