Combining in silico transcriptome mining and biological mass spectrometry for neuropeptide discovery in the Pacific white shrimp Litopenaeus vannamei

• Published in: Peptides (New York, N.Y. : 1980) Vol. 31; no. 1; pp. 27 - 43
• Main Authors: Ma, Mingming, Gard, Ashley L., Xiang, Feng, Wang, Junhua, Davoodian, Naveed, Lenz, Petra H., Malecha, Spencer R., Christie, Andrew E., Li, Lingjun
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.01.2010; Elsevier
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; Computational Biology - methods; Computer programs; Databases, Genetic; Electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS); Expressed sequence tag (EST); Expressed Sequence Tags; Foundations; Functional genomics; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Humans; Litopenaeus vannamei; Marine; Mass Spectrometry; Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); Molecular Sequence Data; Neuropeptides - chemistry; Neuropeptides - genetics; Penaeidae - anatomy & histology; Penaeidae - chemistry; Penaeidae - genetics; Peptides; Precursors; Sequence Alignment; Shrimps; Tachykinins - chemistry; Tachykinins - genetics; Vertebrates: endocrinology; Electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS); Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); Functional genomics; Expressed sequence tag (EST); Fourier transform mass spectrometry; MALDI-FTMS; Flight; Matrix-assisted laser desorption/ionization; Neuropeptide; Mass spectrometry
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2009.10.007

The shrimp Litopenaeus vannamei is arguably the most important aquacultured crustacean, being the subject of a multi-billion dollar industry worldwide. To extend our knowledge of peptidergic control in this species, we conducted an investigation combining transcriptomics and mass spectrometry to identify its neuropeptides. Specifically, in silico searches of the L. vannamei EST database were conducted to identify putative prepro-hormone-encoding transcripts, with the mature peptides contained within the deduced precursors predicted via online software programs and homology to known isoforms. MALDI-FT mass spectrometry was used to screen tissue fragments and extracts via accurate mass measurements for the predicted peptides, as well as for known ones from other species. ESI-Q-TOF tandem mass spectrometry was used to de novo sequence peptides from tissue extracts. In total 120 peptides were characterized using this combined approach, including 5 identified both by transcriptomics and by mass spectrometry (e.g. pQTFQYSRGWTNamide, Arg7-corazonin, and pQDLDHVFLRFamide, a myosuppressin), 49 predicted via transcriptomics only (e.g. pQIRYHQCYFNPISCF and pQIRYHQCYFIPVSCF, two C-type allatostatins, and RYLPT, authentic proctolin), and 66 identified solely by mass spectrometry (e.g. the orcokinin NFDEIDRAGMGFA). While some of the characterized peptides were known L. vannamei isoforms (e.g. the pyrokinins DFAFSPRLamide and ADFAFNPRLamide), most were novel, either for this species (e.g. pEGFYSQRYamide, an RYamide) or in general (e.g. the tachykinin-related peptides APAGFLGMRamide, APSGFNGMRamide and APSGFLDMRamide). Collectively, our data not only expand greatly the number of known L. vannamei neuropeptides, but also provide a foundation for future investigations of the physiological roles played by them in this commercially important species.