Partial reduction and two-step modification of proteins for identification of disulfide bonds.
An experimental protocol was established to combine partial reduction, cyanylation, and a second modification step for the assignment of disulfide bonds in proteins that are resistant to proteolysis under native conditions. After proteolysis, disulfide bonds were assigned via MALDI mass spectrometry with subsequent semiautomatic interpretation using the program SearchXLinks, which enumerates all possible combinations of proteolytic fragments for all observed monoisotopic masses. The putative assignment of disulfide bonds was confirmed by ISD and PSD fragmentation of the corresponding protonated molecules.
Schnaible
Volker
V
Center of Advanced European Studies and Research, Stiftung caesar, Bonn, Germany. schnaible@caesar.de
Wefing
Stephan
S
Bücker
Anne
A
Wolf-Kümmeth
Sybille
S
Hoffmann
Daniel
D
D000465:Algorithms; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D010446:Peptide Fragments; D011506:Proteins; D012259:Ribonuclease, Pancreatic; D010447:Peptide Hydrolases