Chiral amino acid analysis for the detection of D-proline in hypertrehalosaemic neuropeptides of cicadas

Malte Bayer, Simone König, Heather Marco, Gerd Gäde

Abstract

For more than two decades, it has been known that the neurosecretory glands of the cicadas, the corpora cardiaca (CC), synthesize two isobaric peptides with hypertrehalosaemic activity. Both decapeptides have the same amino acid sequence (pGlu -Val- Asn -Phe -Ser -Pro -Ser -Trp -Gly -Asn -NH2) but differ in retention time in reversed-phase liquid chromatography. A synthetic peptide with the above sequence co-eluted with the second, more hydrophobic peptide peak of the natural cicada CC extract. The modification associated with the less-hydrophobic peptide was not clear. Ion mobility separation, which is sensitive to changes in conformation, in conjunction with high-resolution mass spectrometry (nanoUPLC, Synapt G2 Si, Waters Corp.), was used to investigate this phenomenon in Platypleura capensis. Different drift times in buffer gas for both the intact peptides and some of their fragment ions were detected. Based on the ion mobility and fragment ion intensity of the corresponding ions it was shown that the region Pro6-Ser7-Trp8 contained a different structural feature to that of the L-amino acids present in the known peptide [1]. The chromatographic behavior of synthetic peptides excluded the presence of D-Ser and D-Trp in these positions, whereas the synthetic peptide containing D-Pro had the same retention time as the less hydrophobic natural peptide [2,3]. We have employed acid hydrolysis and derivatisation of the resulting amino acids with a variant of Marfey's reagent [4] in order to further investigate the presence of D-proline in the unknown peptide hormone