Mass Spectrometric Profiling of Neuropeptides in Response to Copper Toxicity via Isobaric Tagging

Copper is a necessary nutrient but quickly becomes toxic at elevated levels. To properly handle environmental copper influxes and maintain metal homeostasis, organisms utilize various methods to chelate, excrete, and metabolize heavy metals. These mechanisms are believed to involve complex signaling pathways mediated by neuropeptides. This study incorporates custom N,N-dimethyl leucine isobaric tags to characterize the neuropeptidomic changes after different time points (1, 2, and 4 h) of copper exposure in a model organism, blue crab, Callinectes sapidus. Using a modified simplex optimization strategy, the number of identifiable and quantifiable neuropeptides was increased 3-fold to facilitate a deeper understanding of the signaling pathways involved in responding to heavy metal exposure. The time course exposure showed many interesting findings, including upregulation of inhibitory allatostatin peptides in the pericardial organs. Additionally, there was evidence of transport of a pigment dispersing hormone from the sinus glands to the brain. Overall, this study improves the multiplexing capabilities of neuropeptidomic studies to understand the temporal changes associated with copper toxicity.