Amino Acids

, Volume 39, Issue 3, pp 751–761 | Cite as

Shotgun strategy-based proteome profiling analysis on the head of silkworm Bombyx mori

  • Jianying Li
  • S. Hossein Hosseini Moghaddam
  • Xiang Chen
  • Ming Chen
  • Boxiong ZhongEmail author
Original Article


Insect head is comprised of important sensory systems to communicate with internal and external environment and endocrine organs such as brain and corpus allatum to regulate insect growth and development. To comprehensively understand how all these components act and interact within the head, it is necessary to investigate their molecular basis at protein level. Here, the spectra of peptides digested from silkworm larval heads were obtained from liquid chromatography tandem mass spectrometry (LC–MS/MS) and were analyzed by bioinformatics methods. Totally, 539 proteins with a low false discovery rate (FDR) were identified by searching against an in-house database with SEQUEST and X!Tandem algorithms followed by trans-proteomic pipeline (TPP) validation. Forty-three proteins had the theoretical isoelectric point (pI) greater than 10 which were too difficult to separate by two-dimensional gel electrophoresis (2-DE). Four chemosensory proteins, one odorant-binding protein, two diapause-related proteins, and a lot of cuticle proteins, interestingly including pupal cuticle proteins were identified. The proteins involved in nervous system development, stress response, apoptosis and so forth were related to the physiological status of head. Pathway analysis revealed that many proteins were highly homologous with the human proteins which involved in human neurodegenerative disease pathways, probably implying a symptom of the forthcoming metamorphosis of silkworm. These data and the analysis methods were expected to be of benefit to the proteomics research of silkworm and other insects.


Bombyx mori Insect head Proteomics LTQ-Orbitrap Gene Ontology Pathway 



This work was supported by the National Basic Research Program of China (Grant No. 2005CB121003), National Hi-Tech Research and Development Program of China (Grant No. 2006AA10A118), and Program for New Century Excellent Talents in University. We are thankful to the 985-Institute of Agrobiology and Environmental Sciences of Zhejiang University, for providing convenience for our experiment. We are also grateful to Mr. Wei Fan and Jisheng Li for kind help in our data analysis.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary material

726_2010_517_MOESM1_ESM.pdf (150 kb)
Supplementary material 1 (PDF 150 kb)
726_2010_517_MOESM2_ESM.pdf (51 kb)
Supplementary material 2 (PDF 51 kb)
726_2010_517_MOESM3_ESM.xls (1.6 mb)
Supplementary material 3 (XLS 1688 kb)
726_2010_517_MOESM4_ESM.xls (202 kb)
Supplementary material 4 (XLS 201 kb)


  1. Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M (2006) The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol 7:R80CrossRefPubMedGoogle Scholar
  2. Berg D, Holzmann C, Riess O (2003) 14-3-3 Proteins in the nervous system. Nat Rev Neurosci 4:752–762CrossRefPubMedGoogle Scholar
  3. Bier E (2005) Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet 6:9–23CrossRefPubMedGoogle Scholar
  4. Boston PF, Jackson P, Thompson RJ (1982) Human 14-3-3 protein: radioimmunoassay, tissue distribution, and cerebrospinal fluid levels in patients with neurological disorders. J Neurochem 38:1475–1482CrossRefPubMedGoogle Scholar
  5. Dahanukar A, Hallem EA, Carlson JR (2005) Insect chemoreception. Curr Opin Neurobiol 15:423–430CrossRefPubMedGoogle Scholar
  6. de Souza GA, Godoy LM, Mann M (2006) Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol 7:R72CrossRefPubMedGoogle Scholar
  7. Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4:207–214CrossRefPubMedGoogle Scholar
  8. Foret S, Maleszka R (2006) Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome Res 16:1401–1413CrossRefGoogle Scholar
  9. Gong DP, Zhang HJ, Zhao P, Lin Y, Xia QY, Xiang ZH (2007) Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori. Insect Biochem Mol Biol 37:266–277CrossRefPubMedGoogle Scholar
  10. Gorman M, Kuroda MI, Baker BS (1993) Regulation of the sex-specific binding of the maleless dosage compensation protein to the male X chromosome in Drosophila. Cell 72:39–49CrossRefPubMedGoogle Scholar
  11. Greenspan RJ, Dierick HA (2004) ‘Am not I a fly like thee?’—From genes in fruit flies to behavior in humans. Hum Mol Genet 13:R267–R273CrossRefPubMedGoogle Scholar
  12. Grimaldi DA, Engel MS (2005) Evolution of the insects. Cambridge University Press, LondonGoogle Scholar
  13. Gu SH, Chow YS (2005) Analysis of ecdysteroidogenic activity of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori. Arch Insect Biochem Physiol 58:17–26CrossRefPubMedGoogle Scholar
  14. Heller M, Ye M, Michel PE, Morier P, Stalder D, Jünger MA, Aebersold R, Reymond F, Rossier JS (2005) Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides. J Proteome Res 4:2273–2282CrossRefPubMedGoogle Scholar
  15. Hosseini Moghaddam SH, Du X, Li J, Cao JR, Zhong BX, Chen YY (2008) Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure. Biotechnol Bioprocess Eng 13:624–631CrossRefGoogle Scholar
  16. Hsu YC, Chern JJ, Cai Y, Liu M, Choi KW (2007) Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 445:785–788CrossRefPubMedGoogle Scholar
  17. Huang G, Lu H, Hao A, Ng DC, Ponnia S, Guo K, Lufei C, Zeng Q, Cao X (2004) GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I. Mol Cell Biol 24:8447–8456CrossRefPubMedGoogle Scholar
  18. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, Finn RD, Gough J, Haft D, Hulo N, Kahn D, Kelly E, Laugraud A, Letunic I, Lonsdale D, Lopez R, Madera M, Maslen J, McAnulla C, McDowall J, Mistry J, Mitchell A, Mulder N, Natale D, Orengo C, Quinn AF, Selengut JD, Sigrist CJ, Thimma M, Thomas PD, Valentin F, Wilson D, Wu CH, Yeats C (2009) InterPro: the integrative protein signature database. Nucleic Acids Res 37:D211–D215CrossRefPubMedGoogle Scholar
  19. International Silkworm Genome Consortium (2008) The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol 38:1036–1045CrossRefGoogle Scholar
  20. Isobe M, Kai H, Kurahashi T, Suwan S, Pitchayawasin-Thapphasaraphong S, Franz T, Tani N, Higashi K, Nishida H (2006) The molecular mechanism of the termination of insect diapause: Part 1. A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein. Chembiochem 7:1590–1598CrossRefPubMedGoogle Scholar
  21. Itoh MT, Hattori A, Nomura T, Sumi Y, Suzuki T (1995) Melatonin and arylalkylamine N-acetyltransferase activity in the silkworm, Bombyx mori. Mol Cell Endocrinol 115:59–64CrossRefPubMedGoogle Scholar
  22. Kai H, Kotani Y, Miao Y, Azuma M (1995) Time interval measuring enzyme for resumption of embryonic development in the silkworm, Bombyx mori. J Insect Physiol 41:905–910CrossRefGoogle Scholar
  23. Kalume DE, Okulate M, Zhong J, Reddy R, Suresh S, Deshpande N, Kumar N, Pandey A (2005a) A proteomic analysis of salivary glands of female Anopheles gambiae mosquito. Proteomics 5:3765–3777CrossRefPubMedGoogle Scholar
  24. Kalume DE, Peri S, Reddy R, Zhong J, Okulate M, Kumar N, Pandey A (2005b) Genome annotation of Anopheles gambiae using mass spectrometry-derived data. BMC Genomics 6:128CrossRefPubMedGoogle Scholar
  25. Kaneko Y, Takaki K, Iwami M, Sakurai S (2006) Developmental profile of annexin IX and its possible role in programmed cell death of the Bombyx mori anterior silk gland. Zool Sci 23:533–542CrossRefPubMedGoogle Scholar
  26. Keller A, Eng J, Zhang N, Li XJ, Aebersold R (2005) A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol Syst Biol 1:2005.0017CrossRefPubMedGoogle Scholar
  27. Li JY, Chen X, Fan W, Hosseini Moghaddam SH, Chen M, Zhou ZH, Yang HJ, Chen JE, Zhong BX (2009a) Proteomic and bioinformatic analysis on endocrine organs of domesticated silkworm, Bombyx mori L. for a comprehensive understanding of their roles and relations. J Proteome Res 8:2620–2632CrossRefPubMedGoogle Scholar
  28. Li JY, Chen X, Hosseini Moghaddam SH, Chen M, Wei H, Zhong BX (2009b) Shotgun proteomics approach to characterizing the embryonic proteome of silkworm, Bombyx mori, at labrum appearance stage. Insect Mol Biol 18:649–660CrossRefPubMedGoogle Scholar
  29. Mulder N, Apweiler R (2007) InterPro and InterProScan: tools for protein sequence classification and comparison. Methods Mol Biol 396:59–70CrossRefPubMedGoogle Scholar
  30. Nakato H, Izumi S, Tomino S (1992) Structure and expression of gene coding for a pupal cuticle protein of Bombyx mori. Biochim Biophys Acta 1132:161–167PubMedGoogle Scholar
  31. Pandey A, Mann M (2000) Proteomics to study genes and genomes. Nature 405:837–846CrossRefPubMedGoogle Scholar
  32. Rauch A, Bellew M, Eng J, Fitzgibbon M, Holzman T, Hussey P, Igra M, Maclean B, Lin CW, Detter A, Fang R, Faca V, Gafken P, Zhang H, Whiteaker J, States D, Hanash S, Paulovich A, McIntosh MW (2006) Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and high throughput biological experiments. J Proteome Res 5:112–121CrossRefPubMedGoogle Scholar
  33. Rebers JF, Riddiford LM (1988) Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J Mol Biol 203:411–423CrossRefPubMedGoogle Scholar
  34. Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin JF, Bruni AC, Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, Polinsky RJ, Wasco W, Da Silva HAR, Haines JL, Pericak-Vance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St George-Hyslop PH (1995) Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease. Nature 375:754–760CrossRefPubMedGoogle Scholar
  35. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefPubMedGoogle Scholar
  36. Tani N, Kamada G, Ochiai K, Isobe M, Suwan S, Kai H (2001) Carbohydrate moiety of time-interval measuring enzyme regulates time measurement through its interaction with time-holding peptide PIN. J Biochem 129:221–227PubMedGoogle Scholar
  37. Taraszka JA, Kurulugama R, Sowell RA, Valentine SJ, Koeniger SL, Arnold RJ, Miller DF, Kaufman TC, Clemmer DE (2005) Mapping the proteome of Drosophila melanogaster: analysis of embryos and adult heads by LC-IMS-MS methods. J Proteome Res 4:1223–1237CrossRefPubMedGoogle Scholar
  38. Waku Y (1991) Developmental changes of the antenna and its neurons in the silkworm, Bombyx mori, with special regard to larval–pupal transformation. J Morphol 207:253–271CrossRefGoogle Scholar
  39. Wei ZJ, Zhang QR, Kang L, Xu WH, Denlinger DL (2005) Molecular characterization and expression of prothoracicotropic hormone during development and pupal diapause in the cotton bollworm, Helicoverpa armigera. J Insect Physiol 51:691–700CrossRefPubMedGoogle Scholar
  40. Xia Q, Cheng D, Duan J, Wang G, Cheng T, Zha X, Liu C, Zhao P, Dai F, Zhang Z, He N, Zhang L, Xiang Z (2007) Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biol 8:R162CrossRefPubMedGoogle Scholar
  41. Yamashita O (1996) Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function. J Insect Physiol 42:669–679CrossRefGoogle Scholar
  42. Yang P, Tanaka H, Kuwano E, Suzuki K (2008) A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: Cloning and expression pattern in pharate first instar larvae in relation to diapause. J Insect Physiol 54:636–643CrossRefPubMedGoogle Scholar
  43. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293–W297CrossRefPubMedGoogle Scholar
  44. Zhou ZH, Yang HJ, Chen M, Lou CF, Zhang YZ, Chen KP, Wang Y, Yu ML, Yu F, Li JY, Zhong BX (2008) Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet. J Proteome Res 7:5103–5111CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Jianying Li
    • 1
  • S. Hossein Hosseini Moghaddam
    • 1
    • 2
  • Xiang Chen
    • 3
  • Ming Chen
    • 3
  • Boxiong Zhong
    • 1
    Email author
  1. 1.College of Animal SciencesZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.Agriculture FacultyUniversity of GuilanRashtIran
  3. 3.College of Life SciencesZhejiang UniversityHangzhouPeople’s Republic of China

Personalised recommendations