Capture and identification of folding intermediates of cystinyl proteins by cyanylation and mass spectrometry.
Trapping folding intermediates of cystinyl proteins by covalent modification of free sulfhydryl groups provides the opportunity for isolation, purification, and structural elucidation of individual species. The disulfide structure of the intermediates, coupled with their temporal abundance, provides a 'snapshot' of the pathway experienced by the refolding protein in a particular medium. Here, intermediates of cystinyl proteins containing free cysteines are trapped by cyanylation through reaction with an acidic (pH 3.0) solution of 1-cyano-4-dimethylamino-pyridinium (CDAP) tetrafluoroborate. The cyanylated species are separated by reversed-phase high-performance liquid chromatography, where the resulting chromatogram gives a visual indication of the distribution of intermediates at a designated time after commencing the refolding process. The disulfide structure of an intermediate can be determined by cleaving its cyanylated derivative and by mass mapping of the resulting fragments to the sequence of the original protein. Cleavage of a cyanylated species represented by any given peak in the chromatogram is achieved by treatment of that fraction with 1M NH4OH at room temperature for 1 h; the resulting fragments are analyzed by matrix-assisted laser desorption ionization (MALDI) or electrospray mass spectrometry. Examples will be presented from in vitro refolding experiments with human epidermal growth factor (hEGF), for which more than 10 folding intermediates were isolated and identified at different time points, and a mutant of insulin-like growth factor-I, for which three intermediates were isolated and identified.
Watson
J T
JT
Departments of Biochemistry and Chemistry, Michigan State University, East Lansing, MI, USA. watsonj@msu.edu
Yang
Y
Y
Wu
J
J
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000737:chemistry* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009570:Nitriles / Q000737:chemistry; D010084:Oxidation-Reduction; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011726:Pyridinium Compounds / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C084339:LR(3)IGF-I; D009570:Nitriles; D011506:Proteins; D011726:Pyridinium Compounds; D003553:Cystine; C067292:1-cyano-4-dimethylaminopyridinium; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I