Discovery and characterization of Melanoplus sanguinipes AKH II by combined HPLC and mass spectrometry methods.

Reversed-phase high-performance liquid chromatography (RP-HPLC) allows for fractionation of extract from the corpora cardiaca of insects for examination of bioactivity or immunoreactivity and/or subsequent characterization of the peptides. Using RP-HPLC, we have previously identified an adipokinetic hormone (AKH) from the corpora cardiaca of the migratory grasshopper Melanoplus sanguinipes. This hydrophobic decapeptide has the same primary structure as locust (Locusta migratoria; Lom) AKH I. Initially, only one HPLC peak with the same retention time as Lom-AKH I was detected; however, we found a second putative AKH within this fraction by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and micro HPLC tandem electrospray ionization mass spectrometry. Armed with this information, we were able to improve the resolution of the native AKHs by micro HPLC, which allowed us to characterize the peptide. Our results show the power of coupling HPLC and mass spectrometric methods to resolve and identify very similar hydrophobic peptides at the lower picomole level.