Measurement of neuropeptides in crustacean hemolymph via MALDI mass spectrometry

Abstract

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. (J Am Soc Mass Spectrom 2009, 20, 708 -718)

Mass spectrometric measurement of neuropeptide secretion in the crab, Cancer borealis, by in vivo microdialysis

“…It is worth noticing that only 7 out of the 22 NPs also have decent matches with Mascot database searching, which may indicate that PEAKS is more suitable for identification of smaller NPs. As a result of improved instrumentation, more NPs were identified compared to a previous study using a similar sample preparation procedure with a modest resolution MALDI TOF/TOF instrument 12 . Out of these identified NPs, only CCAP and I/LNFTHKFa were detected in both studies.…”

“…Its presence in the decapod crustacean nervous system has been revealed by a number of MS studies 7, 24, 41 . Its presence in circulating hemolymph has also been confirmed 12 . Its canonical function is to stimulate cardiac activity 45 , although it is also active in the STNS 46 .…”

“…These methods include sampling NPs from stimulated neuronal releasate 10, 11 , blood or hemolymph 12 . Direct analysis of signaling neuromodulators without sacrificing the animal via fluid-based methods also allows the study of biologically active molecules under different physiological conditions in a single animal.…”

“…Liquid chromatography (LC) –MS is well-suited to this purpose. Over the last two decades, biological MS has shown powerful capabilities in the discovery of NPs in crustacean neuronal tissues 4, 6, 7, 10, 12 . However, compared with tissue-based NP studies, fluid-based sampling methods coupled with MS for detection of NP release in crustaceans are still poorly developed.…”

Sources and range of long-term variability of rhythmic motor patternsin vivo.

“…Curiously, however, despite stimulation of descending projection neurons both in vitro (Nusbaum and Beenhakker, 2002) and in vivo (Diehl et al, 2013;Hedrich et al, 2011) resulting in clear changes of CPG activity, most reported in vivo data show only the canonical motor patterns typically observed in unperturbed in vitro preparations (Hedrich et al, 2011;Massabuau and Meyrand, 1996;Clemens et al, 1998b). This is in spite of there being clear changes in the STG motor output in vitro Marder and Calabrese, 1996) and in vivo (Heinzel, 1988) in response to application of modulators known to be present in the blood (Li et al, 2002;Chen et al, 2009). It is unclear how prevalent in vivo modulation actually is, how it affects the long-term activity of the STG motor patterns and if the same variability of motor output reported in vitro exists under natural conditions.…”