Visualization and Identification of Neurotransmitters in Crustacean Brain via Multifaceted Mass Spectrometric Approaches

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has emerged as a label-free analytical tool for fast biomolecule profiling on tissue sections. Among various functional molecules, mapping neurotransmitters and related metabolites is of tremendous significance, as these compounds are critical to signaling in the central nervous system. Here, we demonstrated the use of both derivatization and reaction-free approaches that greatly reduced signal complexity and thus enabled complementary signaling molecule visualization on crab brain sections via MALDI-LTQ-Orbitrap XL platform. Pyrylium salt served as a primary amine derivatization reagent and produced prominent signal enhancement of multiple neurotransmitters, including dopamine, serotonin, γ-aminobutyric acid, and histamine that were not detected in underivatized tissues. Molecules with other functional groups, such as acetylcholine and phosphocholine, were directly imaged after matrix application. The identities of discovered neurotransmitters were verified by standards using LC-MS/MS. This study broadens our understanding of metabolic signaling in the crustacean nervous system and highlights potential of multifaceted MS techniques for unambiguous neurotransmitter characterization.