Comparación de métodos de extracción de proteínas de cerebro y linfocitos de rata

Comparison of protein extraction methods from brain and lymphocytes of rat

  • Karen Maldonado-Moreno UACJ. Dpto. de Ciencias Químico Biológicas. Instituto de Ciencias Biomédicas
  • Rocío Martell-Gaytán UACJ. Dpto. de Ciencias Químico Biológicas. Instituto de Ciencias Biomédicas
  • Bonifacio Alvarado-Tenorio UACJ. Dpto. de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas https://orcid.org/0000-0002-4591-1807
  • José Valero-Galván UACJ. Dpto. de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas https://orcid.org/0000-0002-2752-6761
  • Alejandro Martínez-Martínez UACJ. Dpto. de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas https://orcid.org/0000-0003-3448-910X
  • Ángel Gabriel Díaz-Sánchez UACJ. Dpto. de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas https://orcid.org/0000-0002-6398-7274
  • Raquel González-Fernández UACJ. Dpto. de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas
Palabras clave: proteómica, electroforesis bidimensional (2-DE), extracción de proteínas, cerebro, linfocitos

Resumen

En este trabajo se compararon tres técnicas de extracción de proteínas actualmente empleadas en proteómica, para determinar la más eficiente para realizar electroforesis bidimensional (2-DE) en tejido cerebral y linfocitos de sangre periférica de rata. Los métodos utilizados fueron el uso directo de solución de lisis, el método TCA/acetona-DTT y el método TCA/acetona-fenol. Una vez que se realizó la extracción, se separaron las proteínas por medio de electroforesis en geles de poliacrilamida en condiciones desnaturalizantes (SDS-PAGE) y 2-DE, con el objetivo de seleccionar cuál de ellos brindó un mayor rendimiento en la cantidad de proteínas totales, así como en el número de bandas bien definidas y manchas bien enfocadas en los geles 2-DE, tanto para cerebro como para linfocitos. Al comparar el perfil proteico, en cerebro se detectaron 13 ± 0; 15 ± 1 y 12 ± 1 bandas bien definidas mediante los métodos de TCA/ acetona-DTT, TCA/acetona-fenol y solución de lisis, respectivamente. En linfocitos, se encontraron 19 ± 1.20 ± 0 y 19 ± 1 bandas, respectivamente. Con respecto al proteoma, tanto en cerebro como en linfocitos se encontró mayor número de manchas proteicas consistentes y bien enfocadas con el método de TCA/acetona-DTT. Estos resultados mostraron que el mejor método de extracción de proteínas para su uso en la 2-DE correspondió al de TCA/acetona-DTT, siendo además más rápido y sencillo de realizar que el método de TCA/acetona-fenol.

DOI: https://doi.org/10.54167/tch.v11i3.87

Citas

Alam, M. & W. Ghosh. 2014. Optimization of a phenol extraction-based protein preparation method amenable to downstream 2DE and MALDI-MS based analysis of bacterial proteomes. Proteomics 14(2-3): 216-221. https://doi.org/10.1002/pmic.201300146

Bradford, M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analylical Biochemistry 72(1-2): 248-254. https://doi.org/10.1016/0003-2697(76)90527-3

Cilia, M., T. Fish, X. Yang, M. Mclaughlin, T.W. Thannhauser & S. Gray. 2009. A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins. J. Biomol. Tech. 20(4): 201-215. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729484/

Deatherage, B.L., D.S. Wunschel, M.A. Sydor, M.G. Warner, K.L. Wahl & J.R. Hutchison. 2015. Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins. Journal of Microbiological Methods 118:18-24. https://doi.org/10.1016/j.mimet.2015.08.008

Ericsson, C., I. Peredo & M. Nistér. 2007. Optimized protein extraction from cryopreserved brain tissue samples. Acta Oncológica 46(1): 10-20. https://doi.org/10.1080/02841860600847061

Faurobert, M., E. Pelpoir & J. Chaïb. 2007. Phenol extraction of proteins for proteomic studies of recalcitrant plant tissues. En Plant Proteomics. Methods and Protocols (pp.9-14). Humana Press. ISBN 9781071605271.

Gao, M., N. Li, J. Zhang, P. Yang & X. Zhang. 2006. The study of three extraction methods for pre-separation and enrichment: Application to the complex proteome separation in rat liver. Separation and Purification Technology 52(1):170-176. https://doi.org/10.1016/j.seppur.2006.04.006

Glatter, T., E. Ahrné & A. Schmidt. 2015. Comparison of different sample preparation protocols reveals lysis buffer-specific extraction biases in gram-negative bacteria and human cells. Journal of Proteome Research 14(11): 4472-4485. https://doi.org/10.1021/acs.jproteome.5b00654

González, R., J. Valero & J.V. Jorrín-Novo. 2014. Proteómica en hongos fitopatógenos. En Manual de Proteómica (pp.585-607). Sociedad Española de Proteómica. https://tinyurl.com/4ff9t93v

Görg, A., W. Postel, S. Gunther & C. Friedrich. 1988. Horizontal two-dimensional electrophoresis with immobilized pH gradients using PhastSystem. Electrophoresis 9(1): 57-59. https://doi.org/10.1002/elps.1150090111

Hao, R., C. Adoligbe, B. Jiang, X. Zhao, L. Gui, K. Qu, S. Wu & L. Zan. 2015. An optimized trichloroacetic acid/acetone precipitation method for two-dimensional gel electrophoresis analysis of qinchuan cattle longissimus dorsi muscle containing high proportion of marbling. PLoS ONE 10(4): 1-12. https://doi.org/10.1371/journal.pone.0124723

Haudenschild, D.R., A. Eldridge, P. J. Lein & B. A. Chromy. 2014. High abundant protein removal from rodent blood for biomarker discovery. Biochemical and Biophysical Research Communications 455(1-2): 84-89. https://doi.org/10.1016/j.bbrc.2014.09.137

Herosimczyk, A., N. Dejeans, T. Sayd, M. Ozgo, W.E. Skrzypczak & A. Mazur. 2006. Plasma proteome analysis: 2D gels and chips. Journal of Physiology and Pharmacology 57(7):81-97. https://tinyurl.com/yc8nfckw

Jankowska, U., A. Latosinska, B. Skupien-Rabian, B. Swiderska, M. Dziedzicka-Wasylewska & S. Kedracka-Krok. 2016. Optimized procedure of extraction, purification and proteomic analysis of nuclear proteins from mouse brain. Journal of Neuroscience Methods 261(1):1-9. https://doi.org/10.1016/j.jneumeth.2015.12.002

Kim, M. & C. Kim. 2007. Human blood plasma preparation for two-dimensional gel electrophoresis. Journal of Chromatography 849(1-2): 203-210. https://doi.org/10.1016/j.jchromb.2006.11.046

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680-5.

Liley, K.S., A. Razzaq & P. Dupree. 2002. Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Current Opinion in Chemical Biology 6(1): 46-50. https://doi.org/10.1016/S1367-5931(01)00275-7

Luche, S., V. Santoni & T. Rabilloud. 2003. Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3(3): 249-253. https://doi.org/10.1002/pmic.200390037

Malafaia, C.B., M. L. Guerra, T. D. Silva, P. M.G. Paiva, E. B. Souza, M. T. S. Correia & M. V. Silva. 2015. Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach. Proteome Science 13(5). https://proteomesci.biomedcentral.com/articles/10.1186/s12953-015-0062-9

Maldonado, A., S. Echeverría-Zomeño, S. Jean-Baptiste, M. Hernández & J.V. Jorrín-Novo. 2008. Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis. J. Proteomics 71(4): 461-472. https://doi.org/10.1016/j.jprot.2008.06.012

Moore, S.M., S.M. Hess & J.W. Jorgenson. 2016. Extraction, enrichment, solubilization, and digestion techniques for membrane proteomics. Journal of Proteome Research 15(4):1243-1252. https://doi.org/10.1021/acs.jproteome.5b01122

Northrop, R.B. & A.N. Connor. 2009. Introduction to molecular biology, genomics and proteomics for biomedical engineers. CRC Press. ISBN 9780429148767. https://doi.org/10.1201/b15770

Pooladi, M., S.K.R. Abad & M. Hashemi. 2014. Proteomics analysis of human brain glial cell proteome by 2D gel. Indian Journal of Cancer 51(2): 159-162. https://www.indianjcancer.com/text.asp?2014/51/2/159/138271

Rabilloud, T., M. Chevallet, S. Luche & C. Lelong. 2010. Two-dimensional gel electrophoresis in proteomics: Past, present and future. Journal of Proteomics. 73(11): 2064-77. https://doi.org/10.1016/j.jprot.2010.05.016

Rabilloud, T. & C. Lelong. 2011. Two-dimensional gel electrophoresis in proteomics: A tutorial. Journal of Proteomics 74(10): 1829-1841. https://doi.org/10.1016/j.jprot.2011.05.040

Sheoran, I.S., A.R.S. Ross, D.J.H. Olson & V. K. Sawhney. 2009. Compatibility of plant protein extraction methods with mass spectrometry for proteome analysis. Plant Science 176(1): 99-104. https://doi.org/10.1016/j.plantsci.2008.09.015

Shevchenko, G., S. Musunuri, M. Wetterhall & J. Bergquist. 2012. Comparison of extraction methods for the comprehensive analysis of mouse brain proteome using shotgun-based mass spectrometry. Journal of Proteome Research 11(4): 2441-2451. https://doi.org/10.1021/pr201169q

Tenório-Daussat, C.L., M.C. Martinho, R.L. Ziolli, R.A. Hauser-Davis, D. Schaumloffel & T. Saint’Pierre. 2014. Evaluation and standardization of different purification procedures for fish bile and liver metallothionein quantification by spectrophotometry and SDS-PAGE analyses. Talanta 120: 491-497. https://doi.org/10.1016/j.talanta.2013.11.070

Tiong, H., S. Hartson & P.M. Muriana. 2015. Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry. Journal of Microbiological Methods 110: 54-60. https://doi.org/10.1016/j.mimet.2015.01.004

Vargas-Caraveo, A., H. Castillo-Michel, G.E. Mejía-Carmona, D.G. Pérez-Ishiwara, M. Cotteb & A. Martínez-Martínez. 2014. Preliminary studies of the effect of psychological stress on circulating lymphocytes analyzed by synchrotron radiation based-Fourier transform infrared microspectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128(15): 141-146. https://doi.org/10.1016/j.saa.2014.02.148

Wang, W., R. Vignani, M. Scali & M. Cresti. 2006. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis 27(13): 2782-2786. https://doi.org/10.1002/elps.200500722

Zheng, Q., J. Song, K. Doncaster, E. Rowland & D. Byers. 2007. Qualitative and quantitative evaluation of protein extraction protocols for apple and strawberry fruit suitable for two-dimensional electrophoresis and mass spectrometry analysis. J. Agricultural and Food Chemistry 55(5): 1663-1673. https://doi.org/10.1021/jf062850p

Publicado
2017-12-15
Cómo citar
Maldonado Moreno, K., Martell Gaytán, R., Alvarado Tenorio, B., Valero Galván, J., Martínez Martínez, A., Díaz-Sánchez, Ángel G., & González-Fernández, R. (2017). Comparación de métodos de extracción de proteínas de cerebro y linfocitos de rata: Comparison of protein extraction methods from brain and lymphocytes of rat. TECNOCIENCIA Chihuahua, 11(3), 127-137. https://doi.org/10.54167/tch.v11i3.87