Discovery and Characterization of the Crustacean Hyperglycemic Hormone Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniques

Abstract

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules—neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, one novel orcomyotropin, one novel Ork/Orcomyotropin-related and one novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides of the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.

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

“…After centrifugation, supernatant fractions were combined and concentrated to dryness. The sample was re-suspended in 100 l of water for further analysis (5). The detailed protocol is described in Supplementary Materials.…”

“…Nano-LC-ESI-QTOF MS/MS was performed using a Waters nanoAcquity UPLC system coupled to a QTOF Micro mass spectrometer (Waters, Milford, MA) as described previously (5). The MS/MS raw data were converted to peak list (.pkl) files using ProteinLynx software 2.4 (Waters) (5). Peptides were identified by searching against an NCBInr 20090726 protein database (9330197 sequences; 3196564765 residues) using the Mascot v2.1 search engine.…”

“…Our group and others have greatly expanded the peptidomes of many model organisms (3, 18 -33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20 -40 members in a simple crustacean model system (5,6,(25)(26)(27)(28)(29)(30)(31)34). However, a majority of these neuropeptides are small peptides with 5-15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome.…”

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

“…The benefit of ETD in bottom-up proteomics has been increasingly documented, whereas endogenous peptides remain largely unexplored by ETD, despite the expectation that ETD would improve sequencing for larger peptides. In the few studies on endogenous peptides (11,12), ETD did not cover large peptides exceeding 5000 Da. Because we have used CID to facilitate the discovery of previously unknown biologically active peptides (3,13,14), we were interested to see if ETD would be helpful to identify endogenous peptides that have escaped identification by CID.…”

Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery

“…Studies can involve endogenous peptides (Hui et al, 2011), peptidomic profiling (Jahn et al, 2011), and de novo sequencing of peptides . Neuropeptidomics focuses on biologically active short segments of peptides and have been investigated in numerous species including Rattus (Dowell et al, 2006;Wei et al, 2006), Mus musculus (Che and Fricker, 2005;Fricker, 2010), Bovine taurus (Colgrave et al, 2011), Japanese quail diencephalon (Scholz et al, 2010), and invertebrates Fu and Li, 2005;Hummon et al, 2006;Vilim et al, 2010).…”