Measurement of neuropeptides in crustacean hemolymph via MALDI mass spectrometry

作者：Ruibing Chen, Mingming Ma, Limei Hui, Jiang Zhang, Lingjun Li · 发表于：Journal of the American Society for Mass Spectrometry · 年份：2008 · DOI：10.1016/j.jasms.2008.12.007 · 被引用次数：55 · 研究领域：Neurobiology and Insect Physiology Research、Invertebrate Immune Response Mechanisms、Mass Spectrometry Techniques and Applications

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 ...