Investigation of signaling molecules and metabolites found in crustacean hemolymph via in vivo microdialysis using a multifaceted mass spectrometric platform

Neurotransmitters (NTs) are endogenous signaling molecules that play an important role in regulating various physiological processes in animals. Detection of these chemical messengers is often challenging due to their low concentration levels and fast degradation rate in vitro. In order to address these challenges, herein we employed in vivo microdialysis (MD) sampling to study NTs in the crustacean model Cancer borealis. Multifaceted separation tools, such as CE and ion mobility mass spectrometry (MS) were utilized in this work. Small molecules were separated by different mechanisms and detected by MALDI mass spectrometric imaging (MALDI-MSI). Performance of this separation-based MSI platform was also compared to LC-ESI-MS. By utilizing both MALDI and ESI-MS, a total of 208 small molecule NTs and metabolites were identified, of which 39 were identified as signaling molecules secreted in vivo. In addition, the inherent property of sub microscale sample consumption using CE enables shorter time of MD sample collection. Temporal resolution of MD was improved by approximately tenfold compared to LC-ESI-MS, indicating the significant advantage of applying separation-assisted MALDI-MS imaging platform.