A robust protein extraction method for two dimensional electrophoresis of silkworm proteins
Affiliation(s)
Institute of Life Sciences, Jiangsu University, Zhenjiang, China. School of Life Science, Sichuan University, Chengdu, China.
Institute of Life Sciences, Jiangsu University, Zhenjiang, China. School of Life Science, Sichuan University, Chengdu, China.
ABSTRACT
Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.
Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.
Cite this paper
Qin, L. , Shi, H. , Xia, H. , Wang, Y. , Chen, L. , Yao, Q. and Chen, K. (2013) A robust protein extraction method for two dimensional electrophoresis of silkworm proteins. Advances in Bioscience and Biotechnology, 4, 584-589. doi: 10.4236/abb.2013.44076.
Qin, L. , Shi, H. , Xia, H. , Wang, Y. , Chen, L. , Yao, Q. and Chen, K. (2013) A robust protein extraction method for two dimensional electrophoresis of silkworm proteins. Advances in Bioscience and Biotechnology, 4, 584-589. doi: 10.4236/abb.2013.44076.
References
[1] Cilia, M., Fish, T., Yang, X., McLaughlin, M., Thannhauser, T.W. and Gray, S. (2009) A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins. Journal of Biomolecular Techniques, 20, 201-215. [2] Diane Barraclough, D.O., William, L. and Tanya, C., (2004) A general method for two-dimensional protein electrophoresis of fruit samples. Postharvest Biology and Technology, 32, 175-181. doi:10.1016/j.postharvbio.2003.11.002 [3] Wang, X., Shi, M., Lu, X., Ma, R., Wu, C., Guo, A., Peng, M. and Tian, W. (2010) A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS. Proteome Science, 8, 35-44. doi:10.1186/1477-5956-8-35 [4] Moscardi, F. (1999) Assessment of the application of baculoviruses for control of Lepidoptera. Annual Review of Entomology, 44, 257-289. doi:10.1146/annurev.ento.44.1.257 [5] Kim, Y., Nandakumar, M.P. and Marten, M.R. (2007) Proteome map of Aspergillus nidulans during osmoadaptation. Fungal Genetics and Biology, 44, 886-895. doi:10.1016/j.fgb.2006.12.001 [6] Liang, Y., Chen, H., Tang, M. and Shen, S. (2007) Proteome analysis of an ectomycorrhizal fungus Boletus edulis under salt shock. Mycological Research, 111, 939-946. doi:10.1016/j.mycres.2007.06.005 [7] Bessede, E., Solecki, O., Sifre, E., Labadi, L. and Megraud, F. (2011) Identification of Campylobacter species and related organisms by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Clinical Microbiology and Infection, Vol. 17, pp. 11011106. [8] Zhou, Z.H., Yang, H.J., Chen, M., Lou, C.F., Zhang, Y.Z., Chen, K.P., Wang, Y., Yu, M.L., Yu, F., Li, J.Y., Zhong, B.X. (2008) Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet. Journal of Proteome Research, 7, 5103-5111. doi:10.1021/pr800383r [9] Jenkins, R.E. and Pennington, S.R. (2001) Arrays for protein expression profiling: Towards a viable alternative to two-dimensional gel electrophoresis? Proteomics, 1, 1329. doi:10.1002/1615-9861(200101)1:1<13::AID-PROT13>3.0.CO;2-J [10] Stadler, F. and Hales, D. (2002) Highly-resolving two-dimensional electrophoresis for the study of insect proteins. Proteomics, 2, 1347-1353. doi:10.1002/1615-9861(200209)2:9<1347::AID-PROT1347>3.0.CO;2-P [11] Molloy, M.P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Analytical Biochemistry, 280, 1-10. doi:10.1006/abio.2000.4514 [12] Jiang, L., He, L. and Fountoulakis, M. (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography, 1023, 317-320. doi:10.1016/j.chroma.2003.10.029 [13] Wang Yuqi, P.X. (2006) A two-dimensional electrophoresis protocol suitable for proteomic study of rice leaves. Journal of Plant Physiology and Molecular Biology, 2, 252-256. [14] Cohen, A.M., Rumpel, K., Coombs, G.H. and Wastling, J.M. (2002) Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: Proteomics of Toxoplasma gondii. International Journal for Parasitology, 32, 39-51. doi:10.1016/S0020-7519(01)00308-3
[1] Cilia, M., Fish, T., Yang, X., McLaughlin, M., Thannhauser, T.W. and Gray, S. (2009) A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins. Journal of Biomolecular Techniques, 20, 201-215. [2] Diane Barraclough, D.O., William, L. and Tanya, C., (2004) A general method for two-dimensional protein electrophoresis of fruit samples. Postharvest Biology and Technology, 32, 175-181. doi:10.1016/j.postharvbio.2003.11.002 [3] Wang, X., Shi, M., Lu, X., Ma, R., Wu, C., Guo, A., Peng, M. and Tian, W. (2010) A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS. Proteome Science, 8, 35-44. doi:10.1186/1477-5956-8-35 [4] Moscardi, F. (1999) Assessment of the application of baculoviruses for control of Lepidoptera. Annual Review of Entomology, 44, 257-289. doi:10.1146/annurev.ento.44.1.257 [5] Kim, Y., Nandakumar, M.P. and Marten, M.R. (2007) Proteome map of Aspergillus nidulans during osmoadaptation. Fungal Genetics and Biology, 44, 886-895. doi:10.1016/j.fgb.2006.12.001 [6] Liang, Y., Chen, H., Tang, M. and Shen, S. (2007) Proteome analysis of an ectomycorrhizal fungus Boletus edulis under salt shock. Mycological Research, 111, 939-946. doi:10.1016/j.mycres.2007.06.005 [7] Bessede, E., Solecki, O., Sifre, E., Labadi, L. and Megraud, F. (2011) Identification of Campylobacter species and related organisms by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Clinical Microbiology and Infection, Vol. 17, pp. 11011106. [8] Zhou, Z.H., Yang, H.J., Chen, M., Lou, C.F., Zhang, Y.Z., Chen, K.P., Wang, Y., Yu, M.L., Yu, F., Li, J.Y., Zhong, B.X. (2008) Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet. Journal of Proteome Research, 7, 5103-5111. doi:10.1021/pr800383r [9] Jenkins, R.E. and Pennington, S.R. (2001) Arrays for protein expression profiling: Towards a viable alternative to two-dimensional gel electrophoresis? Proteomics, 1, 1329. doi:10.1002/1615-9861(200101)1:1<13::AID-PROT13>3.0.CO;2-J [10] Stadler, F. and Hales, D. (2002) Highly-resolving two-dimensional electrophoresis for the study of insect proteins. Proteomics, 2, 1347-1353. doi:10.1002/1615-9861(200209)2:9<1347::AID-PROT1347>3.0.CO;2-P [11] Molloy, M.P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Analytical Biochemistry, 280, 1-10. doi:10.1006/abio.2000.4514 [12] Jiang, L., He, L. and Fountoulakis, M. (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography, 1023, 317-320. doi:10.1016/j.chroma.2003.10.029 [13] Wang Yuqi, P.X. (2006) A two-dimensional electrophoresis protocol suitable for proteomic study of rice leaves. Journal of Plant Physiology and Molecular Biology, 2, 252-256. [14] Cohen, A.M., Rumpel, K., Coombs, G.H. and Wastling, J.M. (2002) Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: Proteomics of Toxoplasma gondii. International Journal for Parasitology, 32, 39-51. doi:10.1016/S0020-7519(01)00308-3