title: Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge.
abstract: The tobacco hornworm, Manduca sexta, has been used as a biochemical model for studying insect physiological processes. While the transcriptomes of its fat body, hemocytes, midgut, and antennae have been examined in several studies, limited information is available for proteins in tissues, cells, or body fluids of this insect. In keeping pace with the M. sexta genome project, we launched a pilot study to identify differences in the peptidome of cell-free hemolymph samples from larvae injected with buffer or a mixture of bacteria. At 24 h after injection, plasma was collected and treated with 50% acetonitrile to precipitate large proteins. The supernatants, containing peptides (<25 kDa) and other stable proteins (>25 kDa), were digested with trypsin and analyzed by nano-liquid chromatography and nano-electrospray tandem mass spectrometry (nanoLC-MS/MS) on an LTQ Orbitrap XL mass spectrometer. Known M. sexta cDNA sequences and gene transcripts from the draft genome were translated in silico to generate a database of polypeptides (i.e. peptides and proteins) in this species. By searching the database, we identified 268 hemolymph polypeptides, 50 of which showed 1.67-200 fold abundance increases after the immune challenge, as judged by significant changes in normalized spectral counts between the control and induced plasma. These included a total of 33 antimicrobial peptides (attacins, cecropins, defensins, diapausins, gallerimycin, gloverin, lebocins, lysozymes), pattern recognition receptors, and proteinase inhibitors. Although there was no strong parallel (correlation coefficients: -0.13, 0.11, 0.39 and 0.62) between plasma peptide levels and their transcript levels in control or induced hemocytes or fat body, we observed the mRNA level changes in hemocytes and fat body concurred with their peptide level changes with correlation coefficients of 0.67 and 0.76, respectively. These data suggest that fat body contributed a significant portion of the plasma polypeptides involved in various aspects of innate immunity after the bacterial injection.
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Shuguang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Cao
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Hartson
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA. Electronic address: haobo.jiang@okstate.edu.
authors.forename: Haobo
authors.initials: H
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007113:Immunity, Innate; D019476:Insect Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D007814:Larva / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000382:microbiology; D018402:Manduca / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D059467:Transcriptome
chemical_list: D019476:Insect Proteins; D010455:Peptides
keywords: Antimicrobial peptide; Hemolymph protein; Insect immunity; Spectral counting; Tobacco hornworm; nanoLC-MS/MS