Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating
hormones and regulate many physiological processes. However, the detection of these low-level
peptide hormones in circulation is often complicated by high
salt interference and rapid degradation of
proteins and
peptides in crude hemolymph extracts. In this study, we systematically evaluated three different
neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of
neuropeptides by combining
acid-induced abundant
protein precipitation/depletion, ultrafiltration, and C(18) micro-column desalting. In hemolymph samples collected from the crab
Cancer borealis, several secreted
neuropeptides have been detected, including members from at least five
neuropeptide families, such as
RFamide,
allatostatin,
orcokinin,
tachykinin-related
peptide (TRP), and
crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these
neuropeptides in feeding regulation. In addition, a novel
peptide with a
Lys-Phe-
amide C-terminus was identified and de novo sequenced directly from the
Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant
peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for
neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous
neuropeptides were detected, including both known ones and new
peptides whose functions remain to be characterized.