Single-cell peptidomics of drosophila melanogaster neurons identified by Gal4-driven fluorescence.

Neuropeptides are widespread signal molecules that display a great chemical and functional diversity. Predictions of neuropeptide cleavage from precursor proteins are not always correct, and thus, biochemical identification is essential. Single-cell analysis is valuable to identify peptides processed from a single precursor, but also to determine coexpression of further neuropeptides from other precursors. We have developed an approach to isolate single identified neurons from the fruit fly Drosophila melanogaster for mass spectrometric analysis. By using Gal4 promoter lines to drive green fluorescent protein under UAS control, we identified specific peptidergic neurons. These neurons were isolated singly under a fluorescence microscope and subjected to MALDI-TOF mass spectrometry. Two Gal4 lines were used here to identify pigment-dispersing factor (PDF) and hugin-expressing neurons. We found that the large PDF expressing clock neurons only give rise to a single peptide, PDF. The three different classes of hugin expressing neurons all display the same mass signal, identical to pyrokinin-2. The other peptide predicted from the hugin precursor, hugin gamma, was not detected in any of the cells. Single-cell peptidomics is a powerful tool in Drosophila neuroscience since Gal4 drivers can be produced for all known neuropeptide genes and thus provide detailed information about neuropeptide complements in neurons of interest.