DOI QR코드

DOI QR Code

Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

  • Lee, Jae Eun ;
  • Lee, Jae Young ;
  • Kim, Hong Rye ;
  • Shin, Hyun Young ;
  • Lin, Tao ;
  • Jin, Dong Il
  • Received : 2014.10.11
  • Accepted : 2015.01.19
  • Published : 2015.06.01

Abstract

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

Keywords

2D DIGE;CyDye;Proteomics;Bovine Pregnancy;Pregnancy-specific Serum Proteins

References

  1. Anderson, N. G., A. Matheson, and N. L. Anderson. 2001. Back to the future: the human protein index (HPI) and the agenda for post-proteomic biology. Proteomics 1:3-12. https://doi.org/10.1002/1615-9861(200101)1:1<3::AID-PROT3>3.0.CO;2-T
  2. Alban, A., S. O. David, L. Bjorkesten, C. Andersson, E. Sloge, S. Lewis, and I. Currie. 2003. A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3:36-44 https://doi.org/10.1002/pmic.200390006
  3. Bartol, F. F., R. M. Roberts, F. W. Bazer, and W. W. Thatcher. 1985. Characterization of proteins produced in vitro by bovine endometrial explants. Biol. Reprod. 33:745-759. https://doi.org/10.1095/biolreprod33.3.745
  4. Berendt, F. J., T. Frohlich, S. E. Schmidt, H. D. Reichenbach, E. Wolf, and G. J. Arnold. 2005. Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period. Proteomics 5:2551-2560. https://doi.org/10.1002/pmic.200401242
  5. Bichsel, V. E., L. A. Liotta, and E. F. Petricoin III. 2001. Cancer proteomics: from biomarker discovery to signal pathway profiling. Cancer J. 7:69-78
  6. Butler, J. E., W. C. Hamilton, R. G. Sasser, C. A. Ruder, G. M. Hass, and R. J. Williams. 1982. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol. Reprod. 26:925-933. https://doi.org/10.1095/biolreprod26.5.925
  7. Chromy, B. A., A. D. Gonzales, J. Perkins, M. W. Choi, M. H. Corzett, B. C. Chang, C. H. Corzett, and S. L. McCutchen-Maloney. 2004. Proteomic analysis of human serum by twodimensional differential gel electrophoresis after depletion of high-abundant proteins. J. Proteome Res. 3:1120-1127. https://doi.org/10.1021/pr049921p
  8. Gharbi, S., P. Gaffney, A. Yang, M. J. Zvelebil, R. Cramer, M. D. Waterfield, and J. F. Timms. 2002. Evaluation of twodimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol. Cell. Proteomics 1:91-98. https://doi.org/10.1074/mcp.T100007-MCP200
  9. Gorg, A., C. Obermaier, G. Boguth, A. Harder, B. Scheibe, R. Wildgruber, and W. Weiss. 2000. The current state of twodimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037-1053. https://doi.org/10.1002/(SICI)1522-2683(20000401)21:6<1037::AID-ELPS1037>3.0.CO;2-V
  10. Hochstrasser, D. F., S. Frutiger, M. R. Wilkins, G. Hughes, and J. C. Sanchez. 1997. Elevation of apolipoprotein E in the CSF of cattle affected by BSE. FEBS Lett. 416:161-163. https://doi.org/10.1016/S0014-5793(97)01191-5
  11. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. Collections and ficolins: humoral lectins of the innate immune defense. Ann. Rev. Immunol. 21:547-578. https://doi.org/10.1146/annurev.immunol.21.120601.140954
  12. Knickerboker, J. J., W. W. Thatcher, F. W. Bazer, M. Drost, D. H. Barron, K. B. Fincher, and R. M. Roberts. 1986. Proteins secreted by day-16 to -18 bovine conceptuses extend corpus luteum function in cows. J. Reprod. Fertil. 77:381-391. https://doi.org/10.1530/jrf.0.0770381
  13. Manabe, T., Y. Takahashi, and T. Okuyama. 1987. Identification of bovine fetal and adult serum/plasma proteins by twodimensional electrophoresis and immunochemical staining. Electrophoresis 8:573-579. https://doi.org/10.1002/elps.1150081207
  14. Marouga, R., S. David, and E. Hawkins. 2005. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal. Bioanal. Chem. 382:669-678. https://doi.org/10.1007/s00216-005-3126-3
  15. Morita, A., E. Miyagi, H. Yasumitsu, H. Kawasaki, H. Hirano, and F. Hirahara. 2006. Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma. Proteomics 6:5880-5890. https://doi.org/10.1002/pmic.200500708
  16. Nation, D. P., J. Malmo, G, M. Davis, and K. L. Macmillan. 2003. Accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. Aust. Vet. J. 81:63-65. https://doi.org/10.1111/j.1751-0813.2003.tb11435.x
  17. Ndimba, B. K., S. Chivasa, W. J. Simon, and A. R. Slabas. 2005. Identification of Arabidopsis salt and osmotic stress responsive proteins using two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 5:4185-4196. https://doi.org/10.1002/pmic.200401282
  18. Oltenacu, P. A., J. D. Ferguson, and A. J. Lednor. 1990. Economic evaluation of pregnancy diagnosis in dairy cattle: A decision analysis approach. J. Dairy Sci. 73:2826-2831. https://doi.org/10.3168/jds.S0022-0302(90)78970-9
  19. Perenyi, Z. S., O. Szenci, J. Sulon, P. V. Drion, and J. F. Beckers. 2002. Comparison of the ability of three radioimmunoassay to detect pregnancy-associated glycoproteins in bovine plasma. Reprod. Domest. Anim. 37:100-104. https://doi.org/10.1046/j.1439-0531.2002.00341.x
  20. Pyo, J., S. I. Hwang, J. Oh, S. J. Lee, S. C. Kang, J. S. Kim, and J. Lim. 2003. Characterization of a bovine pregnancy-associated protein using two-dimensional gel electrophoresis, N-terminal sequencing and mass spectrometry. Proteomics 3:2420-2427. https://doi.org/10.1002/pmic.200300618
  21. Raggiaschi, R., C. Lorenzetto, E. Diodato, A. Caricasole, S. Gotta, and G. C. Terstappen. 2006. Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology. Proteomics 6:748-756. https://doi.org/10.1002/pmic.200500064
  22. Sasser, R. G., C. A. Ruder, K. A. Ivani, J. E. Butler, and W. C. Hamilton. 1986. Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol. Reprod. 35: 936-942. https://doi.org/10.1095/biolreprod35.4.936
  23. Sasser, R. G., J. Crock, and C. A. Ruder-Montgomery. 1989. Characteristics of pregnancy-specific protein B in cattle. J. Reprod. Fertil. Suppl. 37:109-113.
  24. Talamo, F., C. D'Ambrosio, S. Arena, P. D. Vecchio, L. Ledda, G. Zehender, L. Ferrara, and A. Scaloni. 2003. Proteins from bovine tissues and biological fluids: Defining a reference electrophoresis map for liver, kidney, muscle, plasma and red blood cells. Proteomics 3:440-460. https://doi.org/10.1002/pmic.200390059
  25. Tong, R., J. Shaw, B. Middleton, R. Rowlinson, S. Rayner, J. Young, F. Pognan, E. Hawkins, I. Currie, and M. Davision. 2001. Validation and development of fluorescence twodimensional differential gel electrophoresis proteomics technology. Proteomics 1:377-396. https://doi.org/10.1002/1615-9861(200103)1:3<377::AID-PROT377>3.0.CO;2-6
  26. Unlu, M., M. E. Morgan, and J. S. Minden. 1997. Difference gel electrophoresis. A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071-2077. https://doi.org/10.1002/elps.1150181133
  27. Yan, J. X., A. T. Devenish, R. Wait, T. Stone, S. Lewis, and S. Fowler. 2002. Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli. Proteomics 2:1682-1698. https://doi.org/10.1002/1615-9861(200212)2:12<1682::AID-PROT1682>3.0.CO;2-Y
  28. Yuan, C., Y. Guo, R. Ravi, K. Przyklenk, N. Shilkofsk, R. Diez, R. N. Cole, and A. M. Murphy. 2006. Myosin binding protein C is differentially phosphorylated upon myocardial stunning in canine and rat hearts-evidence for novel phosphorylation sites. Proteomics 6:4176-4186. https://doi.org/10.1002/pmic.200500894
  29. Zhou, G., H. Li, D. DeCamp, S. Chen, H. Shu, Y. Gong, M. Flaig, J. W. Gillespie, N. Hu, P. R. Taylor, M. R. Emmert-Buck, L. A. Liotta, E. F. 3rd. Petricoin, and Y. Zhao. 2002. 2D differential In-gel electrophoresis for the identification of Esophageal Scans Cell cancer-specific protein markers. Mol. Cell. Proteomics 1:117-123. https://doi.org/10.1074/mcp.M100015-MCP200

Cited by

  1. Challenges and opportunities of bovine milk analysis by mass spectrometry vol.13, pp.1, 2016, https://doi.org/10.1186/s12014-016-9110-4
  2. Identification of serum protein markers for early diagnosis of pregnancy in buffalo vol.88, pp.8, 2016, https://doi.org/10.1111/asj.12754
  3. The different expressed serum proteins in rhCygb treated rat model of liver fibrosis by the optimized two-dimensional gel electrophoresis vol.12, pp.6, 2017, https://doi.org/10.1371/journal.pone.0177968

Acknowledgement

Supported by : Rural Development Administration, Ministry for Agriculture, Food and Rural Affairs