Temporal Study of the Perturbation of Crustacean Neuropeptides Due to Severe Hypoxia Using 4-Plex Reductive Dimethylation

Hypoxia (i.e., low oxygen (O₂) levels) is a common environmental challenge for several aquatic species, including fish and invertebrates. To survive or escape these conditions, these animals have developed novel biological mechanisms, some regulated by neuropeptides. By utilizing mass spectrometry (MS), this study aims to provide a global perspective of neuropeptides in the blue crab, Callinectes sapidus, and their changes over time (0, 1, 4, and 8 h) due to acute, severe hypoxia (∼10% O₂ water saturation) stress using a 4-plex reductive dimethylation strategy to increase throughput. Using both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) MS, this study provides complementary coverage, allowing 88 neuropeptides to be identified. Interesting trends include (1) an overall decrease in neuropeptide expression due to hypoxia exposure, (2) a return to basal levels after 4 or 8 h of exposure following an initial response, (3) changes only after 4+ h exposure, and (4) an oscillating pattern. Overall, this study boosts the power of multiplexed quantitation to understand the large-scale changes due to severe hypoxia stress over time.