Abstract
Copper is an essential nutrient with critical roles in protein structure and enzyme activity but
becomes toxic at elevated levels due to its ability to participate in redox reactions. Copper
toxicity is a growing concern as environmental copper concentrations increase due to
agricultural and industrial runoff and poorly managed wastewater. The dysregulation of copper
homeostasis has been linked to many diseases including anemia, liver damage, and
neurodegenerative diseases, but the exact mechanisms are not fully understood. The
biochemical response to copper toxicity has been shown to involve neuropeptides, but the full
suite of relevant neuropeptides and their expression changes are difficult to characterize due to
the complexity of the nervous system. Crustaceans provide a highly relevant model organism
with a simple, well-characterized nervous system, facilitating meaningful analysis of
environmental stress. Neuropeptides remain challenging to study, however, due to their
structural diversity and low in vivo concentrations. As a result, mass spectrometry (MS) has
become the preferred method of analyzing neuropeptides. MS is highly sensitive and selective,
capable of providing both structural and quantitative information, and requires no prior
knowledge of the analytes in the sample. This enables the analyses of not only neuropeptides,
but also the chelating molecules responsible for maintaining homeostasis, such as glutathione
and metallothionein proteins. To thoroughly probe the biochemical and physiological response
to copper toxicity, I propose to: 1) develop and optimize high-throughput, multiplexed mass
spectrometry techniques to quantify neuropeptides, 2) globally profile the neuropeptides
involved in the stress response to both chronic and acute copper toxicity, and 3) analyze the
expression changes of copper chelating molecules in the gills, hemolymph, and hepatopancreas
using top-down MS methods. The proposed research will provide a deeper understanding of the
signaling molecules and biochemical defense mechanisms involved in responding to excess
copper, thereby providing a better understanding of how environmental stressors (such as
heavy metals) can have profound impacts on health. Moreover, understanding the stress
response can provide unique insights into the many diseases linked to copper toxicity and
dysregulation of copper homeostasis.
