High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

Abstract

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and posttranslational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on largescale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone ( Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4 -6).Crustacean hyperglycemic hormone (CHH) 1 family neuropeptides play a central role in energy homeostasis of crustaceans (7-17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs)

Transcriptome profiling of the eyestalk of precocious juvenile Chinese mitten crab reveals putative neuropeptides and differentially expressed genes

“…The chromatophorotropins include two peptides: PDH and PCH. The rapid expansion of crustacean sequencing data and development of bioinformatics in recent years has increased the number of putative neuropeptide sequences from different crustacean species (Christie et al, 2008;Jia et al, 2012;Stewart et al, 2013;Ventura et al, 2014). These neuropeptides are essential for crustacean growth and postembryonic development, including molting and regeneration, and have long been the primary focus of research into improving crustacean aquaculture (Jung et al, 2012).…”

A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system

“…A 0.5 μL of peptide sample was injected onto an Ultimate 3000 RSLCnano system coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) for data-dependent MS/MS analysis with electron-transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) according to our previous report [29]. …”

“…In a previous study [29], we established a strategy by combining bottom-up, off-line top-down, and on-line top-down MS methods for confident de novo sequencing of crustacean hyperglycemic hormone (CHH)-family neuropeptides with molecular weight (MW) around 8.4-9.2 kDa. The current work aims to discover and confidently identify various sizes of novel neuropeptides (MW 0.9-8.2 kDa) in the crustacean nervous system.…”

Mass spectrometric analysis of spatio-temporal dynamics of crustacean neuropeptides

“…Larger neuropeptides from the CHH superfamily with molecular weights higher than 8 kDa were spatially characterized using MS imaging in the sinus gland (SG) of both C. borealis and C. sapidus for the first time by Jia et al [93]. As shown in Figure 5, the distribution patterns of the members from two different CHH subfamilies were reported to be in stark contrast, indicating their distinct signaling pathways in the physiological process.…”