A novel methodology for assignment of disulfide bond pairings in proteins.
A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.
Wu
J
J
Department of Chemistry, Michigan State University, East Lansing 48824, USA.
Watson
J T
JT
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry; D007536:Isomerism; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010720:Phosphines / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010720:Phosphines; D011506:Proteins; D013438:Sulfhydryl Compounds; C080938:tris(2-carboxyethyl)phosphine