Trypanosoma cruzi alkaline 2-DE: Optimization and application to comparative proteome analysis of flagellate life stages.
BACKGROUND
Trypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels.


RESULTS
Different IEF conditions using 6-11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2-DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages.


CONCLUSIONS
High resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile.
Magalhães
Adriana D
AD
Department of Cell Biology, University of Brasília, Brazil. admagalhaes@unb.br
Charneau
Sébastien
S
Paba
Jaime
J
Guércio
Rafael A P
RA
Teixeira
Antonio R L
AR
Santana
Jaime M
JM
Sousa
Marcelo V
MV
Ricart
Carlos A O
CA
Electrophoresis. 2000 Nov;21(17):3649-56
11271483
J Mol Evol. 2005 Dec;61(6):742-57
16315106
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W638-44
15215467
Electrophoresis. 1997 Mar-Apr;18(3-4):324-7
9150909
Parasitol Today. 2000 Jul;16(7):282-6
10858646
Proteomics. 2002 Jan;2(1):3-10
11788986
J Proteome Res. 2007 Dec;6(12):4677-83
17949049
Science. 2005 Jul 15;309(5733):404-9
16020724
Electrophoresis. 1999 Apr-May;20(4-5):712-7
10344237
Mol Biochem Parasitol. 2006 Sep;149(1):1-9
16682088
Proteomics. 2002 Feb;2(2):127-34
11840558
Science. 2005 Jul 15;309(5733):473-6
16020736
Proteomics. 2003 May;3(5):580-3
12748938
Mol Biochem Parasitol. 2007 Jul;154(1):6-21
17499861
Mem Inst Oswaldo Cruz. 2003 Jul;98(5):577-91
12973523
Microbiol Rev. 1995 Jun;59(2):223-40
7603410
Science. 2005 Jul 15;309(5733):409-15
16020725
Proteomics. 2004 Apr;4(4):1052-9
15048986
Proteomics. 2003 Feb;3(2):129-38
12601805
Mol Biochem Parasitol. 2006 Apr;146(2):151-62
16442642
Electrophoresis. 1995 Jul;16(7):1079-86
7498150
Proteomics. 2004 Apr;4(4):943-9
15048976
BMC Genomics. 2007 Jun 12;8:163
17565672
Proteomics. 2002 Nov;2(11):1630-2
12442261
Proteomics. 2002 Aug;2(8):1061-3
12203901
Int J Parasitol. 2004 Jul;34(8):881-6
15217726
J Proteome Res. 2004 May-Jun;3(3):517-24
15253433
Proteomics. 2002 Dec;2(12):1672-81
12469337
J Mass Spectrom. 2007 Nov;42(11):1422-32
17960573
Electrophoresis. 2000 Jul;21(13):2617-21
10949138
Trends Parasitol. 2003 Nov;19(11):495-501
14580960
EMBO J. 2002 Apr 15;21(8):1881-8
11953307