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 (cysteine2S3). At that time, studies concerning the chemistry of sulfur-transferring enzyme systems suggested that cysteine2S3 also existed in biological systems. Two decades later, a cystine trisulfide structure was postulated in the regulator protein molecule for the activation of δ-aminolevulinate synthetase. Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys182−Cys189 in human growth hormone. We have detected a trisulfide structure in methionyl human growth hormone in the major loop disulfide Cys53−Cys165. 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 Cys53−Cys165 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.