Isolation and characterization of a succinimide variant of methionyl human growth hormone.
Deamidation of asparagine and glutamine residues, isomerization of aspartic acid side chains, and racemization of the L- to the D-form of the amino acids are common spontaneous chemical reactions known to occur in proteins. Previous studies have implicated succinimides as intermediates in these reactions; however, the evidence has been indirect. Our results demonstrate, for the first time, the presence of a succinimide intermediate in an intact protein. The succinimide (cyclic imide) variant was isolated from thermally stressed recombinant methionyl human growth hormone (hGH) by high performance anion-exchange chromatography, further purified by reversed-phase high performance liquid chromatography, and analyzed by tryptic mapping. A later eluting tryptic peptide, compared with the native T12 peptide (residues 128-134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed by mass spectrometry (MS). This variant had a protonated molecular mass of 755.3 atomic mass units (u), as compared with 773.3 u for the native T12 peptide. A difference of 18 u, a loss of water, is consistent with the formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS analysis of the cyclic imide-containing peptide verified that the modification occurred at Asp-130. A difference of 18 u was also observed for the intact cyclic imide methionyl hGH variant (22,238 u), as measured by electrospray mass spectrometry, compared with native methionyl hGH (22,256 u).
Teshima
G
G
Department of Medicinal and Analytical Chemistry, , Genentech, Inc., South San Francisco, California 94080.
Stults
J T
JT
Ling
V
V
Canova-Davis
E
E
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D013006:Growth Hormone / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*; D014357:Trypsin
D010446:Peptide Fragments; D013388:Succinimides; D001224:Aspartic Acid; D013006:Growth Hormone; D008715:Methionine; D014357:Trypsin