RESEARCH PAPER The hypertrehalosemic neuropeptides of cicadas are structural isomers—evidence by ion mobility mass spectrometry Simone König 1 & Heather Marco 2 & Gerd Gäde 2 Received: 16 June 2017 /Revised: 2 August 2017 /Accepted: 11 August 2017 /Published online: 29 August 2017 # Springer-Verlag GmbH Germany 2017 Abstract It has been known for more than 20 years that the neurosecretory glands of the cicadas, the corpora cardiaca, synthesize two isobaric peptides with hypertrehalosemic ac- tivity. Both decapeptides have exactly the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn- NH 2 ) and mass but differ in their retention time in reversed- phase liquid chromatography. A synthetic peptide with the same sequence elutes together with the second more hydro- phobic peptide peak of the natural cicada extract. It is not clear what modification is causing the described observations. Therefore, in the current study, ion mobility separation in con- junction with high-resolution mass spectrometry was used to investigate this phenomenon as it was sensitive to changes in conformation. It detected different drift times in buffer gas for both the intact peptides and some of their fragment ions. Based on the ion mobility and fragment ion intensity of the corresponding ions, it is concluded that the region Pro 6 -Ser 7 - Trp 8 contains a structural feature differing from the L-amino acids present in the known peptide. Whether the conformer is the result of racemization or other biochemical processes needs to be further investigated. Keywords Ion mobility mass spectrometry . AKH . Conformation . Isomer Abbreviations AKH Adipokinetic hormone DT Drift time IMS Ion mobility separation MALDI Matrix-assisted laser desorption MS Mass spectrometry RP-LC Reversed-phase liquid chromatography RT Retention time Introduction As in vertebrates, peptides represent the largest single class of neuroregulatory substances in insects [1]. These neuropeptides regulate a large variety of processes including molting, growth, ion and water balance, muscle contraction and energy metabo- lism [2, 3]. One of the best known insect neuropeptide families is the so-called adipokinetic hormone (AKH) family involved mainly in metabolic regulation of fat (adipokinetic), carbohy- drate (hypertrehalosemic) and proline (hyperprolinemic) me- tabolism [2, 3]. These blocked octa-, nona- or decapeptides are synthesized in the corpora cardiaca (CC) and have been found in almost every insect order; more than 60 members of this family are structurally known [4, 5]. Post-translational modifications, such as an unusual Trp-glycosylation, hydroxy- proline and phosphorylated and sulphated Thr residues have been shown [4, 6]. For more than 20 years, it is known that various species of cicadas contain two AKHs with hypertrehalosemic activity in their CC [7–9]. Interestingly, the two peptides could be clearly Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-017-0583-4) contains supplementary material, which is available to authorized users. * Simone König koenigs@uni-muenster.de 1 Core Unit Proteomics, Interdisciplinary Center for Clinical Research, University of Münster, Röntgenstr. 21, 48149 Münster, Germany 2 Department of Biological Sciences, University of Cape Town, Rondebosch, Cape Town 7701, South Africa Anal Bioanal Chem (2017) 409:6415–6420 DOI 10.1007/s00216-017-0583-4