[
International Worm Meeting,
2021]
Neuropeptides are a class of bioactive peptides that obtain their biological activity after cleavage from larger precursor proteins. Once released, these peptides are able to regulate standard physiological functions such as digestion and reproduction but also exert long-term effects in more complex processes, including behavioral adaptation, memory processes and aging. Hence, neuropeptides are often an interesting entry point in an effort to study and better understand these types of behavior. Current strategies for studying relative differences in the neuropeptidome often rely on RNA sequencing, which remains completely blind to the real abundancies of actual neuropeptides, as these result from extensive post-translational processing. On the other hand, current mass spectrometric methods for neuropeptide identification are discovery-driven and lack robustness and reproducibility, hampering quantitative studies. We here present a mass spectrometry-based approach for the relative quantification of the neuropeptidome in C. elegans, using a parallel reaction monitoring method. Our current method can detect and quantify 288 mature neuropeptides, this corresponds to 67% of the (known and predicted) neuropeptidome of C. elegans. When applied to wild type controls, our method identified 178 neuropeptides with a 96% overlap between samples. Insofar as we are aware, we hereby are able to provide the most extensive method to map the peptidome of an animal. We are validating the method for differential studies, delivering a promising avenue to finally enable the detection and differential analysis of neuropeptidomic variations over different conditions.
Boonen, Kurt, De Haes, Wouter, Temmerman, Liesbet, Van Bael, Sven, Edwards, Samantha Louise, Schoofs, Liliane
[
International Worm Meeting,
2017]
Neuropeptides constitute a functionally diverse family of neurochemical signaling molecules. C. elegans is well-suited for the study of neuropeptide biochemistry and function, since numerous peptides are known to contribute to many behavioral regimens, yet deleting peptides - or their processing enzymes altogether - does not affect C. elegans viability. We have developed an optimized protocol that enables the detection of about 120 individual peptides per sample (under reference conditions; treatment may increase or decrease this number), as such vastly increasing the detection potential of C. elegans peptidomics. As a proof of concept, we applied this protocol to characterize the C. elegans enzymes required for the last step in the production of many bioactive peptides - the carboxyterminal amidation reaction - via mutant analysis, and faulty carboxyterminal amidation indeed results in a severely altered neuropeptide profile. Overall, about half of the more than 250 predicted peptides have been detected with our current method under standard conditions, and we are working on expanding the potential even further. We estimate the number of currently detectable peptides to be somewhat larger, as it can be expected that several peptides would be produced under specific conditions only, yet some low-abundant peptides that are expressed in only a few cells still escape detection in whole-mount peptidomics. This problem should be solved by our efforts in establishing single-cell peptidomics approaches, which will increase the signal-to-noise ratio drastically.