Trapping of intermediates during the refolding of recombinant human epidermal growth factor (hEGF) by cyanylation, and subsequent structural elucidation by mass spectrometry.
Human epidermal growth factor (hEGF) contains 53 amino acids and three disulfide bonds. The unfolded, reduced hEGF is allowed to refold under mildly alkaline conditions. The folding is quenched at different time points by adjusting the pH to 3.0 with an acetic acid solution of 1-cyano-4-dimethylamino-pyridinium (CDAP) which traps folding intermediates via cyanylation of free sulfhydryl groups. The mixture of cyanylated intermediates is separated by reversed-phase HPLC; the fractions collected are identified by mass spectrometry. The disulfide structures of the intermediates are then determined by specific chemical cleavage and mass-mapping by MALDI-MS, a novel approach developed in our laboratory. The procedure of quenching and trapping of disulfide intermediates in acidic solution minimizes sulfhydryl-disulfide exchange, and therefore provides a good measure of folding kinetics and preservation of intermediate species. Our cyanylation methodology for disulfide mapping is simpler, faster, and more sensitive than the more conventional approach. Among 18 folding intermediates isolated and identified at different time points, disulfide structures of seven well-populated intermediates, including two non-native isomers with scrambled disulfide structures, one 2-disulfide intermediate, and four 1-disulfide intermediates, have been characterized; most of them possess non-native disulfide structures.
Wu
J
J
Department of Chemistry, Michigan State University, East Lansing 48824, USA.
Yang
Y
Y
Watson
J T
JT
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009570:Nitriles / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D017510:Protein Folding; D011726:Pyridinium Compounds / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism
D004220:Disulfides; D009570:Nitriles; D010446:Peptide Fragments; D011726:Pyridinium Compounds; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C067292:1-cyano-4-dimethylaminopyridinium; D004815:Epidermal Growth Factor