J Vet Sci.  2013 Jun;14(2):107-114. 10.4142/jvs.2013.14.2.107.

Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry

• Siddiki AM

• KMID: 1705511
• DOI: http://doi.org/10.4142/jvs.2013.14.2.107

Abstract

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.

Keyword

bioinformatics; Cryptosporidium; proteomics; SDS-PAGE; tandem mass spectrometry

MeSH Terms

Chemical Fractionation/methods
Chromatography, Liquid/methods/veterinary
Cryptosporidium parvum/*chemistry/growth & development/metabolism
Electrophoresis, Polyacrylamide Gel/methods/veterinary
Gene Expression Profiling/*methods/veterinary
Proteome/analysis
Proteomics/*methods
Protozoan Proteins/*analysis
Sporozoites/chemistry/metabolism
Tandem Mass Spectrometry/methods/veterinary
Proteome
Protozoan Proteins

Figure

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  Fig. 1 (A) 1D-SDS-PAGE analysis of sporozoite proteins of Cryptosporidium (C.) parvum. Total no. of oocysts ~ 107. Total amount of protein~60 µg. Electrophoresed proteins were visualized with colloidal coomassie stain. Molecular weight markers (in kilodaltons) are shown on the left. (B) The lane containing C. parvum proteins was excised into 20 slices. Each gel slice was then digested by trypsin and analyzed by LC-MS/MS.

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  Fig. 2 Hit redundancy after LC-MS/MS analysis of different bands of 1D-SDS gel of C. parvum sporozoite protein. Redundant hits include non-Cryptosporidium hits and hits with the same identical peptides, but matching different accession numbers in the protein databases.

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  Fig. 3 Bimodal distribution of 33 Cryptosporidium proteins identified by a MASCOT search of LC-MS/MS data after analysing 1D-SDS-PAGE gel bands. The search was carried out against the NCBInr database. The x-axis shows the predicted isoelectric points (pI) of the proteins on a linear scale and the y-axis shows the predicted molecular weight (Mr) of the proteins on a logarithmic scale. The dotted box indicates proteins potentially amenable to analysis by 2-DE.

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  Fig. 4 Functional categorization of 33 Cryptosporidium proteins identified through a MASCOT search of different bands of 1D-SDS-PAGE gel. Protein categorization was made according to the MIPS functional Catalogue Database.

Reference

1. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science. 2004; 304:441–445.

2. Coombs GH. Biochemical peculiarities and drug targets in Cryptosporidium parvum: lessons from other coccidian parasites. Parasitol Today. 1999; 15:333–338.

3. Denton H, Brown SM, Roberts CW, Alexander J, McDonald V, Thong KW, Coombs GH. Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii. Mol Biochem Parasitol. 1996; 76:23–29.

4. Doolan DL, Southwood S, Freilich DA, Sidney J, Graber NL, Shatney L, Bebris L, Florens L, Dobano C, Witney AA, Appella E, Hoffman SL, Yates JR 3rd, Carucci DJ, Sette A. Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data. Proc Natl Acad Sci U S A. 2003; 100:9952–9957.

5. Entrala E, Mascaró C. Glycolytic enzyme activities in Cryptosporidium parvum oocysts. FEMS Microbiol Lett. 1997; 151:51–57.

6. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ. A proteomic view of the Plasmodium falciparum life cycle. Nature. 2002; 419:520–526.

7. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DMA, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002; 419:498–511.

8. Heiges M, Wang H, Robinson E, Aurrecoechea C, Gao X, Kaluskar N, Rhodes P, Wang S, He CZ, Su Y, Miller J, Kraemer E, Kissinger JC. CryptoDB: a Cryptosporidium bioinformatics resource update. Nucleic Acids Res. 2006; 34:D419–D422.

9. Ivens A. Hurrah for genome projects! Parasitol Today. 2000; 16:317–320.

10. Lasonder E, Ishihama Y, Andersen JS, Vermunt AMW, Pain A, Sauerwein RW, Eling WMC, Hall N, Waters AP, Stunnenberg HG, Mann M. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature. 2002; 419:537–542.

11. O'Donoghue PJ. Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol. 1995; 25:139–195.

12. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999; 20:3551–3567.

13. Sam-Yellowe TY, Florens L, Wang T, Raine JD, Carucci DJ, Sinden R, Yates JR 3rd. Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites. J Proteome Res. 2004; 3:995–1001.

14. Sanderson SJ, Xia D, Prieto H, Yates J, Heiges M, Kissinger JC, Bromley E, Lal K, Sinden RE, Tomley F, Wastling JM. Determining the protein repertoire of Cryptosporidium parvum sporozoites. Proteomics. 2008; 8:1398–1414.

15. Snelling WJ, Lin Q, Moore JE, Millar BC, Tosini F, Pozio E, Dooley JSG, Lowery CJ. Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa). Mol Cell Proteomics. 2007; 6:346–355.

16. Thompson RCA, Olson ME, Zhu G, Enomoto S, Abrahamsen MS, Hijjawi NS. Cryptosporidium and cryptosporidiosis. Adv Parasitol. 2005; 59:77–158.

17. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Edgar R, Federhen S, Geer LY, Helmberg W, Kapustin Y, Kenton DL, Khovayko O, Lipman DJ, Madden TL, Maglott DR, Ostell J, Pruitt KD, Schuler GD, Schriml LM, Sequeira E, Sherry ST, Sirotkin K, Souvorov A, Starchenko G, Suzek TO, Tatusov R, Tatusova TA, Wagner L, Yaschenko E. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2006; 34:D173–D180.

18. Xu P, Widmer G, Wang Y, Ozaki LS, Alves JM, Serrano MG, Puiu D, Manque P, Akiyoshi D, Mackey AJ, Pearson WR, Dear PH, Bankier AT, Peterson DL, Abrahamsen MS, Kapur V, Tzipori S, Buck GA. The genome of Cryptosporidium hominis. Nature. 2004; 431:1107–1112.