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 delta-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.
Medicinal and Analytical Chemistry Department, Genentech, Inc., South San Francisco, California 94080, USA.
E
E
Canova-Davis
I P
IP
Baldonado
R C
RC
Chloupek
V T
VT
Ling
R
R
Gehant
K
K
Olson
B L
BL
Gillece-Castro
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D013006:Growth Hormone / Q000096:biosynthesis / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry
D010455:Peptides; D013440:Sulfides; D013006:Growth Hormone; D008715:Methionine