Distinction between the three disulfide isomers of guanylin 99-115 by low-energy collision-induced dissociation.
Guanylin 99-115 is a small human peptide hormone with two disulfide bonds. The four cysteinyl residues in this peptide allow the formation of two disulfide bridges in three different ways but only the 1-3/2-4 combination is able to bind to the receptor and cause an increase of intracellular cGMP, while the other isomers are biologically inactive. Using guanylin 99-115 as a model peptide, the aim of this study was to investigate whether it is possible to distinguish the differently bridged isomers directly by tandem mass spectrometry. Guanylin isomers were generated by performing an air oxidation of fully reduced guanylin 1-3/2-4 obtained by chemical synthesis. The reaction product is a mixture of guanylin 1-4/2-3 and guanylin 1-2/3-4 in a ratio of 3:1, but there is virtually no guanylin 1-3/2-4. The two biologically inactive peptides were separated by reversed-phase high pressure liquid chromatography (HPLC). Using low-energy collision-induced dissociation tandem mass spectrometry, it was possible to distinguish unambiguously between the three guanylin isomers. This was possible due to the identification of a large number of fragments with intact disulfide bonds. Accordingly, this strategy of a direct and sensitive analysis should work as well for other peptides, with the potential to determine an undefined disulfide bond pattern.
Badock
V
V
Lower Saxony Institute for Peptide Research (IPF), Hannover, Germany.
Raida
M
M
Adermann
K
K
Forssmann
W G
WG
Schrader
M
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D005768:Gastrointestinal Hormones; D006801:Humans; D007536:Isomerism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D045265:Natriuretic Peptides; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004220:Disulfides; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010446:Peptide Fragments; D010455:Peptides; C116390:guanylin 99-115; C072685:guanylin; D006152:Cyclic GMP