title: Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform.
abstract: Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.
authors.affiliation: State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University , Nanjing 210009, China.|School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Ye
authors.forename: Jingxin
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Zichuan
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Chenxi
authors.initials: C
authors.lastname: Jia
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Claire
authors.initials: C
authors.lastname: Schmerberg
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Proteomics Center of Excellence and Chemistry of Life Processes Institute, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Proteomics Center of Excellence and Chemistry of Life Processes Institute, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Proteomics Center of Excellence and Chemistry of Life Processes Institute, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|School of Life Sciences, Tianjin University , No. 92 Weijin Road, Nankai District, Tianjin 300072, China.
authors.forename: Lingjun
authors.initials: L
authors.lastname: Li
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D030562:Databases, Protein; D007447:Invertebrate Hormones / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D033521:Palinuridae / Q000737:chemistry* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation
chemical_list: D007447:Invertebrate Hormones; D009479:Neuropeptides; D021382:Protein Sorting Signals; D020543:Proteome
keywords: Panulirus interruptus; ProSight; de novo sequencing; dimethylation-assisted fragmentation; high-resolution and high-accuracy mass spectrometry; mass spectrometric imaging; neuropeptide; peptidomics