Confirmation by mass spectrometry of a trisulfide variant in methionyl human growth hormone biosynthesized in Escherichia coli

A sulfur-containing compound found in acid hydrolysates of proteins was identified 30 years ago as a trisulfide: bis-(2-amino-2-carboxyethyl) trisulfide (cysteine(2)S(3)). At that time, studies concerning the chemistry of sulfur-transferring enzyme systems suggested that cysteine(2)S(3) also existed in biological systems, Two decades later, a cystine trisulfide structure was postulated in the regulator protein molecule for the activation of delta-aminolevulinate synthetase, Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys(182)-Cys(189) in human growth hormone, Wet have detected a trisulfide structure in methionyl human growth hormone iu the major loop disulfide Cys(53)-Cys(165). The development of mass spectral analyses of high molecular weight molecules, such as proteins, led to the eventual identification of the modification, A tandem mass spectral analysis on a Sciex electrospray instrument localized an addition of 32 Da to the Cys(53)-Cys(165) fragment, Elemental composition was determined by accurate mass measurement obtained by peak matching to a synthetic peptide and established that an extra sulfur atom was involved.