Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass-Spectrometry-Imaging Protocol

Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information from immunohistochemistry with peptidomics information from mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues; however, these studies did not detect neuropeptide complements in manners comparable to those of conventional peptidomics. The aim of our study was to improve sample preparation so that MSI could provide comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 pm spatial resolution, which was confirmed by parallel analysis of tissue extracts using electrospray-ionization MS. Altogether, more than 100 peptide signals from 15 neuropeptide-precursor genes could be traced with high spatial resolution. In addition, MSI spectra confirmed differential prohormone processing and distinct neuropeptide-based compartmentalization of the retrocerebral complex. We believe that our workflow facilitates incorporation of MSI in neuroscience-related topics, including the study of complex neuropeptide interactions within the CNS.