Mass Spectrometry-Based Peptidomics for the Discovery and Profiling of Endogenous Peptides in Crustacean Hemolymph.

Crustacean hemolymph contains a diverse array of bioactive peptides, including antimicrobial peptides (AMPs) and neuropeptides, which regulate essential immune and physiological processes. However, rapid coagulation and the presence of high-abundance proteins complicate mass spectrometry (MS)-based peptidomic analysis. To address these issues, we systematically evaluated five extraction workflows to optimize endogenous peptide recovery from the hemolymph of the American lobster (Homarus americanus) and the blue crab (Callinectes sapidus). Among the tested approaches, Method 3, which combines anticoagulants and protease inhibitors with a 10 kDa molecular-weight-cutoff filtration step, achieved the highest peptide identification rates. This workflow minimized clotting-related peptide loss and enzymatic degradation, resulting in markedly improved recovery of AMPs with diverse hydrophobicity profiles in both species. Notably, omitting the reduction and alkylation steps in Method 3 further enhanced AMP recovery. For example, in C. sapidus, inclusion of these steps (Method 4) led to an approximately 40% decrease in AMP identifications, likely due to increased sample handling and prolonged incubation. Method 3 also maximized neuropeptide recovery in H. americanus, enabling identification of predicted mature orcokinins and myosuppressins. In C. sapidus, neuropeptide recovery varied across workflows, with Method 3 providing competitive performance. Additionally, we report six novel putative neuropeptides in C. sapidus and identify nine and 11 in silico-predicted novel AMPs in C. sapidus and H. americanus, respectively, characterized by distinct amino acid motifs. Collectively, this study establishes an integrated extraction strategy that enables more comprehensive profiling of crustacean hemolymph peptidomes and provides the first combined report of predicted AMPs and neuropeptides in C. sapidus and H. americanus. These findings expand the repertoire of candidate molecules with potential roles in antimicrobial defense and neuromodulation.