Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides.
Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes.
Collins
Christopher J
CJ
Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94143, USA.
Schilling
Birgit
B
Young
Malin
M
Dollinger
Gavin
G
Guy
R Kiplin
RK
D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000138:chemical synthesis / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D010103:Oxygen Isotopes; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D003432:Cross-Linking Reagents; D010103:Oxygen Isotopes; D010455:Peptides; D016222:Fibroblast Growth Factor 2