Remember Me
Or use your Academic/Social account:


Or use your Academic/Social account:


You have just completed your registration at OpenAire.

Before you can login to the site, you will need to activate your account. An e-mail will be sent to you with the proper instructions.


Please note that this site is currently undergoing Beta testing.
Any new content you create is not guaranteed to be present to the final version of the site upon release.

Thank you for your patience,
OpenAire Dev Team.

Close This Message


Verify Password:
Verify E-mail:
*All Fields Are Required.
Please Verify You Are Human:
fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Paul Millares; E James Lacourse; Samirah Perally; Deborah A Ward; Mark C Prescott; Jane E Hodgkinson; Peter M Brophy; Huw H Rees
Publisher: Public Library of Science (PLoS)
Journal: PLoS ONE
Languages: English
Types: Article
Subjects: Toxicology, qx_200, Research Article, Biology, Proteomics, Veterinary Science, Veterinary Diseases, Veterinary Toxicology, Medicine, Agriculture, Q, R, Agrochemicals, Veterinary Parasitology, Biochemistry, Science, Pest Control, Animal Management, qu_58.5
Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This study demonstrates the cost-effective use of available EST databases and inexpensive, accessible MALDI-TOF MS in conjunction with PMF for reliable protein identification in unsequenced organisms.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1. Hunt DF, Yates JR, 3rd, Shabanowitz J, Winston S, Hauer CR (1986) Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci U S A 83: 6233-7.
    • 2. Yates JR, 3rd (1998) Mass spectrometry and the age of the proteome. J Mass Spectrom 33: 1-19.
    • 3. Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60: 2299-301.
    • 4. Pappin DJ, Hojrup P, Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 3: 327-32.
    • 5. Kaplan RM (2004) Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol 20: 477-81.
    • 6. Wolstenholme AJ, Fairweather I, Prichard R, von Samson-Himmelstjerna G, Sangster NC (2004) Drug resistance in veterinary helminths. Trends Parasitol 20: 469-76.
    • 7. Perry BD, Randolph TF (1999) Improving the assessment of the economic impact of parasitic diseases and of their control in production animals. Vet Parasitol 84: 145-68.
    • 8. McKellar QA, Jackson F (2004) Veterinary anthelmintics: old and new. Trends Parasitol 20: 456-61.
    • 9. Jabbar A, Iqbal Z, Kerboeuf D, Muhammad G, Khan MN, et al. (2006) Anthelmintic resistance: the state of play revisited. Life Sci 79: 2413-31.
    • 10. Sackett D, Holmes P, Abbott K, Jephcott S, Barber M (2006) Assessing the economic cost of endemic disease on the profitability of Australian beef cattle and sheep producers. Project AHW.087 Report, Meat and Livestock Australia, North Sidney. pp 1-119.
    • 11. Beech R, Levitt B, Cambosc M, Zhoud S, Forrestere SG (2010) Association of ion-channel genotype and macrocyclic lactone sensitivity traits in Haemonchus contortus. Mol Biochem Parasitol 171: 74-80.
    • 12. Williamson SM, Wolstenholme AJ (2011) P-glycoproteins of Haemonchus contortus: development of real-time PCR assays for gene expression studies. J Helminthol 1: 1-7.
    • 13. Barrett J, Jefferies JR, Brophy PM (2000) Parasite proteomics. Parasitol Today 16: 400-3.
    • 14. Yatsuda AP, Krijgsveld J, Cornelissen AW, Heck AJ, de Vries E (2003) Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition. J Biol Chem 278: 16941-51.
    • 15. Parkinson J, Whitton C, Schmid R, Thomson M, Blaxter M (2004) NEMBASE: a resource for parasitic nematode ESTs. Nucleic Acids Res 32: 427-30.
    • 16. Mitreva M, Zarlenga DS, McCarter JP, Jasmer DP (2007) Parasitic nematodes - from genomes to control. Vet Parasitol 148: 31-42.
    • 17. Wasmuth JD, Blaxter ML (2004) prot4EST: translating expressed sequence tags from neglected genomes. BMC Bioinformatics 5: 187.
    • 18. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551-67.
    • 19. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54.
    • 20. Go¨rg A, Obermaier C, Boguth G, Harder A, Scheibe B, et al. (2000) The current state of 2-DE with immobilized pH gradients. Electrophoresis 21: 1037-53.
    • 21. Chemale G, Morphew R, Moxon JV, Morassuti AL, Lacourse EJ, et al. (2006) Proteomic analysis of glutathione transferases from the liver fluke parasite, Fasciola hepatica. Proteomics 6: 6263-73.
    • 22. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68: 850-8.
    • 23. Vorm O, Roepstorff P, Mann M (1994) Improved Resolution and Very High Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation. Anal Chem 66: 3281-7.
    • 24. Vizca´ıno JA, Coˆte´ R, Reisinger F, Foster JM, Mueller M, et al. (2009) A guide to the Proteomics Identifications Database proteomics data repository. Proteomics 9: 4276-83.
    • 25. Falkner JA, Falkner JW, Andrews PC (2007) ProteomeCommons.org IO Framework: reading and writing multiple proteomics data formats. Bioinformatics 23: 262-3.
    • 26. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-402.
    • 27. Mackey AJ, Haystead TA, Pearson WR (2002) Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences. Mol Cell Proteomics 1: 139-147.
    • 28. Lottaz C, Iseli C, Jongeneel CV, Bucher P (2003) Modeling sequencing errors by combining Hidden Markov models. Bioinformatics 19: 103-12.
    • 29. Parkinson J, Mitreva M, Whitton C, Thomson M, Daub J, et al. (2004) A transcriptomic analysis of the phylum Nematoda. Nat Genet 36: 1259-67.
    • 30. Hewitson JP, Harcus YM, Curwen RS, Dowle AA, Atmadja AK, et al. (2008) The secretome of the filarial parasite, Brugia malayi: proteomic profile of adult excretory-secretory products. Mol Biochem Parasitol 160: 8-21.
    • 31. Robinson MW, Greig R, Beattie KA, Lamont DJ, Connolly B (2007) Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis. Int J Parasitol 37: 139-48.
    • 32. Smith SK, Nisbet AJ, Meikle LI, Inglis NF, Sales J, et al. (2009) Proteomic analysis of excretory/secretory products released by Teladorsagia circumcincta larvae early post-infection. Parasite Immunol 31: 10-9.
    • 33. Chemale G, Perally S, La Course EJ, Prescott MC, Jones LM, et al. (2010) Comparative Proteomic Analysis of Triclabendazole Response in the Liver Fluke Fasciola hepatica. J. Proteome Res 9: 4940-51.
    • 34. Schuller DJ, Liu Q, Kriksunov IA, Campbell AM, Barrett J, et al. (2005) Crystal structure of a new class of glutathione transferase from the model human hookworm nematode Heligmosomoides polygyrus. Proteins 61: 1024-31.
    • 35. van Rossum AJ, Jefferies JR, Rijsewijk FA, LaCourse EJ, Teesdale-Spittle P, et al. (2004) Binding of hematin by a new class of glutathione transferase from the blood-feeding parasitic nematode Haemonchus contortus. Infect Immun 72: 2780-90.
    • 36. James CE, Davey MW (2009) Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans. Int J Parasitol 39: 213-20.
    • 37. Yan F, Xu L, Liu L, Yan R, Song X, Li X (2009) Immunoproteomic analysis of whole proteins from male and female adult Haemonchus contortus. Vet J Epub. pp 1-6.
    • 38. Skuce PJ, Stewart EM, Smith WD, Knox DP (1999) Cloning and characterization of glutamate dehydrogenase (GDH) from the gut of Haemonchus contortus. Parasitology 118: 297-304.
  • No similar publications.

Share - Bookmark

Funded by projects

  • WT

Cite this article