海马组织分区膜质分离后亚蛋白质组分析方法的建立

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (11): 1217-1222,1226.

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海马组织分区膜质分离后亚蛋白质组分析方法的建立

陈亮¹, 杨建明¹, 李晓明¹, 李晓文¹, 高天明^1,2

1. 南方医科大学生理教研室, 广东, 广州, 510515;
2. 南方医科大学解剖学教研室, 广东, 广州, 510515

出版日期:2004-11-20 发布日期:2004-11-20
基金资助:
基金项目:Supported by for National Natural Science Foundation of ChinaOutstanding Young Scientists (No.30125013;303300240) and Foundationfor Outstanding Young Scientists of PLA (01J009);and Cheung KingScholars Program of China.
作者简介:Gao Tian-ming,Tel:86-20-61648216,E-mail:tgao@fimmu.com
通讯作者:Gao Tian-ming,Tel:86-20-61648216,E-mail:tgao@fimmu.com

Two-dimensional electrophoretic separation of subproteomes in adult rat hippocampal subregions after membrane protein enrichment with sequential extraction

CHEN Liang¹, YANG Jian-ming¹, LI Xiao-ming¹, LI Xiao-wen¹, GAO Tian-ming^1,2

1. 南方医科大学生理教研室, 广东, 广州, 510515;
2. 南方医科大学解剖学教研室, 广东, 广州, 510515

Online:2004-11-20 Published:2004-11-20

摘要/Abstract

摘要： 目的建立适合研究膜质分离后海马组织不同分区亚蛋白质组的实验方法。方法显微切割获得成年大鼠海马不同解剖分区,然后按溶解性不同使用顺序抽提法细胞质和细胞膜蛋白,双向电泳进行分离蛋白后,采用与质谱兼容的高敏感银染显示蛋白斑点,经专业图像软件分析处理评价电泳效果。结果海马各个分区组织中膜蛋白得到有效富集,其中海马齿状回富集效率达8倍,图谱中可以显现的总蛋白斑点数目比传统方法增多30%。结论建立了理想的海马组织分区亚蛋白质组图谱,对膜蛋白实现了有效的富集,可以用于研究不同解剖分区在蛋白水平上的差异,为学习记忆、中枢神经缺血缺氧性损伤等多种后继研究提供了平台。

Abstract: Objective To establish a method for analysis of the subproteomes following membrane protein enrichment, so as to investigate the differences in cytosolic and membrane protein/peptide contents among different hippocampal subregions (CA1, CA3 and dentate gyrus) of rats.Methods The hippocampal subregions were isolated by microdissection, and after membrane protein enrichment with protein solubility-based sequential sample extraction, subproteome profiling was accomplished using modified two-dimensional electrophoresis and silver staining method compatible with high-performance mass spectrometry, which displayed cytosolic or membrane proteins/peptides separately. The efficacy of the electrophoresis was evaluated by image analyzing software.Results The membrane proteins were enriched by 8 folds, and around 30% more spots were shown on the integrated dentate gyrus map than on the conventional map obtained by one-step protein extraction. Such improvement could also be seen in protein mapping of CA1 and CA3 regions.Conclusion When analyzing hippocampal subregions sequential extraction and two-dimensional electrophoresis more effectively separate and display enriched membrane protein/peptides, which may play important roles in such cellular events as signal transduction.

中图分类号:

R338.2

陈亮¹, 杨建明¹, 李晓明¹, 李晓文¹, 高天明^1,2. 海马组织分区膜质分离后亚蛋白质组分析方法的建立[J]. 南方医科大学学报, 2004, 24(11): 1217-1222,1226.

CHEN Liang¹, YANG Jian-ming¹, LI Xiao-ming¹, LI Xiao-wen¹, GAO Tian-ming^1,2. Two-dimensional electrophoretic separation of subproteomes in adult rat hippocampal subregions after membrane protein enrichment with sequential extraction[J]. Journal of Southern Medical University, 2004, 24(11): 1217-1222,1226.

参考文献

[1] Ramon Y, Cajal S. Manual de Histology’a normal y te’cnica microgra’fica[J]. Valencia:Aguilar. 1884
[2] Delgado-Garcia JM, Lopez-Bameo J, Serra R, et al. Electrophysiological and functional identification of different neuronal types within the nucleus ambiguous in the cat[J]. Brain Res, 1983, 277(14):231-40.
[3] Grofova I. The identification of striatal and pallidal neurons projecting to substantia nigra:an experimental study by means of retro grade axonal transport of horseradish peroxidase [J]. Brain Res,1975(23), 91:286-91.
[4] Vandesande F, Dierickx K. Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat [J]. Cell Tissue Res,1975, 164(55):153-62.
[5] Nakazawa K, Quirk MC, Chitwood RA, et al. Requirement for hippocampal CA3 NMDA receptors in associative memory recall[J]. Science, 2002, 297(5579):211-8.
[6] Futatsugi A, Kato K, Ogura H, et al. Facilitation of NMDARindependent LTP and spatial learning in mutant mice lackingryanodine receptor type 3[J]. Neuron, 1999, 24(3):701-13.
[7] Pulsinelli W. Pathophysiology of acute ischemic stroke [J]. Lancet,1992, 339(8792):533-6.
[8] Schreiber SS, Baudry M. Selective neuronal vulnerability in the hippocampus——a role for gene expression [J]? Trends Neurosci,1995, 18(10):446-51.
[9] Lavenex P, Steele MA, Jacobs LF. The seasonal pattern of cell proliferation and neuron number in the dentate gyrus of wild adult eastern grey squirrels[J]. Eur J Neurosci, 2000, 12(2):643-8.
[10] Maurer MH, Feldman RE Jr, Flutterer CD, et al. The proteome of neural stem cells from adult rat hippocampus [J]. Proteome Sci,2003, 1(1):4.
[11] Oguri T, Takahata I, Katsuta K, et al. Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis [J]. Proteomics, 2002, 2(6):666-72.
[12] Tsugita A, Kawakami T, Uchida T, et al. Proteome analysis of mouse brain:two-dimensional electrophoresis profiles of tissue proteins during the course of aging [J]. Electrophoresis, 2000, 21 (9):1853-71.
[13] Gong LW, Gao TM, Huang H, et al. Transient forebrain ischemia induces persistent hyperactivity of large conductance Ca2+-activated potassium channels via oxidation modulation in rat hippocampal CA1 pyramidal neurons[J]. Eur J Neurosci, 2002, 15(4):779-83.
[14] Li XM, Li JG, Hu Ping, et al. Changes in single L-type calcium channel currents in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia [J]. Prog Biochem Biophys, 2003,30(5):755-60.
[15] Li JG, Li XM, Hu Ping, et al. Single channel properties of outwardly rectifying chloride currents in CA1 pyramidal neurons of rat hippocampus[J]. Prog Biochem Biophys, 2003, 30(6):874-78.
[16] Molloy MP, Herbert BR, Welsh BJ, et al. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis[J]. Electrophoresis, 1998, 19(5):837-44.
[17] Lehner I, Niehof M, Borlak J. An optimized method for the isolation and identification of membrane proteins [J]. Electrophoresis, 2003(11), 24:1795-808.
[18] Rabilloud T, Valette C, Lawrence JJ. Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension [J].Electrophoresis, 1994, 15(12):1552-8.
[19] Sanchez JC, Rouge V, Pisteur M, et al. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients [J]. Electrophoresis, 1997, 18(3-4):324-7.
[20] Mortz E, Krogh TN, Vorum H, et al. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis[J]. Proteomics, 2001, 1(11):1359-63
[21] Gauss C, Kalkum M, Lowe M, et al. Analysis of the mouse proteome. (I) Brain proteins:separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation[J]. Electrophoresis, 1999, 20(3):575-600.
[22] Krapfenbauer K, Berger M, Lubec G, et al. Changes in the brain protein levels following administration of kainic acid [J].Electrophoresis, 2001, 22(10):2086-91.

海马组织分区膜质分离后亚蛋白质组分析方法的建立

Two-dimensional electrophoretic separation of subproteomes in adult rat hippocampal subregions after membrane protein enrichment with sequential extraction

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