Mass spectrometric assignment of Leu/Ile in neuropeptides from single neurohemal organ preparations of insects

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF–TOF) tandem mass spectrometry has been applied for the first time on an insect/arthropod target, focusing on PVK/CAP2b neuropeptides in the housefly Musca domestica and flesh fly Neobellieria bullata. The peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The confirmation of side-chain fragments which allows assignment of Leu/Ile even from samples as small as neurohemal organs will greatly accelerate the identification of novel neuropeptides that are implicated in the regulation of critical physiological processes in insects. The unnatural Ile analog is 4.5 times more active than the native Leu sequence in a housefly Malpighian tubule fluid secretion assay, which reinforces the caveat that potency values in a biological assay cannot be relied upon to predict the native sequence.