Peptidomics Approaches for the Identification of Bioactive Molecules from .

Huanglongbing (HLB), a deadly citrus disease, is primarily associated with Liberibacter asiaticus (CLas) and spread by the hemipteran insect . Control strategies to combat HLB are urgently needed. In this work, we developed and compared workflows for the extraction of the peptidome, a dynamic set of polypeptides produced by proteolysis and other cellular processes. High-resolution mass spectrometry revealed bias among methods reflecting the physiochemical properties of the peptides: while TCA/acetone-based methods resulted in enrichment of C-terminally amidated peptides, a modification characteristic of bioactive peptides, larger peptides were overrepresented in the aqueous phase of chloroform/methanol extracts, possibly indicative of reduced co-analytical degradation during sample preparation. Parallel reaction monitoring (PRM) was used to validate the structure and upregulation of peptides derived from hemocyanin, a immune system protein, in insects reared on healthy and CLas-infected trees. Mining of the data sets also revealed 122 candidate neuropeptides, including PK/PBAN family neuropeptides and kinins, biostable analogs of which have known insecticidal properties. Taken together, this information yields new, in-depth insights into peptidomics methodology. Additionally, the putative neuropeptides identified may lead to psyllid mortality if applied to or expressed in citrus, consequently blocking the spread of HLB disease in citrus groves.