Monitoring neuropeptides in vivo via microdialysis and mass spectrometry.

Neuropeptides are important signaling molecules that regulate many essential physiological processes. Microdialysis offers a way to sample neuropeptides in vivo. When combined with liquid chromatography-mass spectrometry detection, many known and unknown neuropeptides can be identified from a live organism. This chapter describes sample preparation techniques and general strategies for the mass spectral analysis of neuropeptides collected via microdialysis sampling. Methods for the in vitro microdialysis of a neuropeptide standard as well as the in vivo microdialysis sampling of neuropeptides from a live crab are described.

Combining microdialysis, NanoLC-MS, and MALDI-TOF/TOF to detect neuropeptides secreted in the crab, Cancer borealis.

Microdialysis is a useful technique for sampling neuropeptides in vivo, and decapod crustaceans are important model organisms for studying how these peptides regulate physiological processes. However, to date, no microdialysis procedure has been reported for sampling neuropeptides from crustaceans. Here we report the first application of microdialysis to sample neuropeptides from the hemolymph of the crab, Cancer borealis. Microdialysis probes were implanted into the pericardial region of live crabs, and the resulting dialysates were desalted, concentrated, and analyzed by LC-ESI-QTOF and MALDI-TOF/TOF mass spectrometry. Analysis of in vitro microdialysates of hemolymph revealed more neuropeptides and fewer protein fragments than hemolymph prepared by typical analysis methods. Mass spectra of in vivo dialysates displayed neuropeptides from 10 peptide families, including the RFamide, allatostatin, and orcokinin families. In addition, GAHKNYLRFa, SDRNFLRFa, and TNRNFLRFa were sequenced from hemolymph dialysates. The detection of these neuropeptides in the hemolymph suggests that they are functioning as hormones as well as neuromodulators. In vivo microdialysis offers the capability to further study these and other neuropeptides in crustacean hemolymph, complementing current tissue-based studies and extending our knowledge of hormonal regulation of physiological states.