Optimization of two-dimensional gel electrophoresis for synaptic proteomics

doi:10.3969/j.issn.2095-4344.2014.24.018

Abstract

Abstract:

BACKGROUND: Two-dimensional electrophoresis (2-DE) is one of the most popular methods of protein separation in synaptic proteomic analysis. However, there was no agreed standard on selection of immobilized pH gradients (IPG) strips and concentration of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the research literature on 2-DE of synaptic proteins.
OBJECTIVE: To optimize IPG strips and concentration of SDS-PAGE in 2-DE for synaptic proteomic analysis, and to get high-quality 2-DE maps of synaptic proteins.
METHODS: Rat hippocampal synapses were adopted in this experiment to compare the effect of 2-DE, which were involved with IPG strips with the linear range of pH 5.0-8.0 and pH 3.0-10.0, IPG strips with the linear and nonlinear range of pH 3.0-10.0, as well as single 10% and 12% SDS-PAGE. Meanwhile, a computer-based online search of PubMed (1989/2013) and CNKI (1989/2013) was performed for the articles about 2-DE of synaptic proteins. The selection of IPG strips and concentration of SDS-PAGE in the research literature were summarized and evaluated, and the distribution characteristics of the synaptic proteins in 2-DE maps of IPG strips with various pH range and SDS-PAGE gels with different concentration were summarized.
RESULTS AND CONCLUSION: The results demonstrated that the synaptic proteins were well separated conveniently and obtained high quality 2-DE maps by nonlinear IPG strips range of pH 3.0-10.0 and single 10% SDS-PAGE gels. At the same time, it was also recommended that combination use of IPG strips range of pH 4.0-7.0 and pH 6.0-11.0, as well as linear gradient 9-16% SDS-PAGE gels. They were all suitable for 2-DE of synaptic proteins.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: synapses, two-dimensional gel electrophoresis, proteomics, SDS-PAGE, rats

CLC Number:

R318

Hu Yong-bo, Gao Li, Wang Dan, Zeng Zhong, Xu Chao-yi, Xiao Bing, Luo Fei-fei, Yang Zhi-yong, Zhou Qiang. Optimization of two-dimensional gel electrophoresis for synaptic proteomics[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(24): 3878-3884.

Figures/Tables 2

2.1 大鼠海马突触超微结构大鼠海马匀浆样品在超速离心管中形成了4个条带，位于1.0-1.2 mol/L蔗糖溶液区间的条带为突触组分，随后的透射电镜检测进一步表明该组分确实为海马突触，见图1。 2.2 不同条件下进行的突触蛋白双向电泳图比较实验共进行了两组突触蛋白双向电泳，第一组使用17 cm的pH 3.0-10.0 L、17 cm的pH 5.0-8.0 L IPG胶条，单一浓度 12% SDS-PAGE凝胶，700 μg蛋白质上样量，考马斯亮蓝染色(图2A，B)；第二组使用pH 3.0-10.0 NL、pH 5.0-8.0 L 17 cm IPG胶条，单一浓度10% SDS-PAGE凝胶，120 μg蛋白质上样量，硝酸银染色(图2C，D)。首先，实验对窄谱(pH 5.0-8.0)和宽谱(pH 3.0-10.0) IPG胶条突触双向电泳图进行了比较。从图2中可看出，IPG 胶条两端区域均存在横条纹，但窄谱(pH 5.0-8.0)较宽谱 (pH 3.0-10.0)胶条两端横条纹明显要少些。此外，经双向电泳分析软件PDQuest 2-DE 8.01分析，窄谱(pH 5.0-8.0)胶条(图2D)分离出效蛋白质点1 560±22个，宽谱(pH 3.0- 10.0)IPG胶条(图2C)分离出有效蛋白质点1 370±19个。也就是说，窄谱(pH 5.0-8.0)相对于宽谱(pH 3.0-10.0)胶条，虽然pH范围变窄，但因展示了部分重叠的蛋白质点，蛋白点的数量不但没有下降，反而平均值还增加了13.84%。从电泳图比较中还看到，窄谱(pH 5.0-8.0)胶条双向电泳图谱效果好，蛋白分离充分、蛋白点圆润、背景清晰，更有利于图谱的分析与比较。不足之处是会损失窄谱pH范围外的突触蛋白点。其次，实验对10%与12%两种不同单一SDS-PAGE凝胶浓度对突触蛋白分离的影响进行了比较。可以看出，第二向电泳采用10% SDS-PAGE(图2C，D)和12% SDS-PAGE凝胶(图2A，B)，蛋白点展示的相对分子质量范围更广，蛋白点分离得更充分。10% SDS-PAGE凝胶分离的蛋白相对分子质量在20 000-170 000的范围内，蛋白点可在整张凝胶上充分展开，而12% SDS-PAGE凝胶分离的蛋白分子量主要集中于14 400-66 200狭窄区域内，多数蛋白点紧密聚集于分子量为35 000-66 200间狭小的凝胶区域，难于分辨。最后，实验比较了线性和非线性宽谱(pH 3.0-10.0) IPG胶条对突触蛋白分离的影响。从图2A，C的比较中不难看出，突触蛋白质等电点大多数还是集中于中段pH范围(pH 5.0-9.0)内，使用非线性pH 3-10 IPG胶条，较线性胶条更能充分展开等电点处于中段pH范围内的突触蛋白。 2.3 突触蛋白双向电泳文献对IPG胶条和SDS-PAGE浓度的选择通过突触蛋白双向电泳相关文献查询，在PubMed数据库中查询到外文文献255篇，在CNKI数据库中查询到中文文献27篇，经阅读，筛选出以突触(排除突触亚组分：突触膜、突触后致密物和突触囊泡)为样品，进行IEF/SDS-PAGE双向电泳的文献有14篇。从表1中可看出，对于IPG胶条的选择，绝大多数实验选择的是pH 3.0-10.0NL文章11篇[4, 15-24]，少数选择了pH 3.0-10.0 L及pH 3.0-11.0 NL各1篇[7，25]，还有1篇文献是合并使用两根pH值相重叠的窄谱IPG胶条(pH 4.0-7.0/pH 6.0-11.0 L)[26]；而对于SDS-PAGE凝胶浓度，选择单一浓度的，5篇选择的是10%[4, 16, 18, 21, 23]，2篇选择11%[15, 25]、1篇选择12.5%[17]，1篇选择13%[26]；选择线性梯度浓度的3篇选择的是9%-16%[19-20, 22]，1篇为8%-18%[24]，1篇为11%-17%[7]。"

2.4 不同浓度SDS-PAGE凝胶对突触蛋白分离能力的比较根据Bio-Rad公司实验操作指南(Ready Gel System RESOURCE GUIDE)上标准蛋白在不同浓度SDS-PAGE凝胶上电泳迁移分布图(图3A)，可见不同浓度(10%，12%，8%-16%，10%-20%)的SDS-PAGE凝胶对相对分子质量为0-116 000的蛋白的分离能力是不同的，10%凝胶对这些蛋白的分离最充分，但会损失大量的0-21 500的小相对分子质量蛋白；8%-16%凝胶的分离能力要大于10%-20%凝胶，尤其是对相对分子质量0-66 000的蛋白。实际的突触蛋白双向电泳图也符合这一规律。实验从实验及文献中共选出了3个代表性浓度(10%，9%-16%，11%-17%)SDS-PAGE凝胶的突触蛋白双向电泳图，实验的10% SDS-PAGE凝胶(图2C，3B)，Buonocore等[22]的9%-16%(接近于8%-16%)SDS-PAGE凝胶(图3C)和Witzmann等[7]的11%-17%(接近于10%-20%)SDS-PAGE凝胶(图3D)。从3张电泳图的比较可发现，10% SDS- PAGE凝胶损失了大量0-21 500的小相对分子质量蛋白，但对于相对分子质量21 500-116 000(尤其是相对分子质量 21 500-66 000)的蛋白比9%-16%和11%-17% SDS- PAGE凝胶分离得更充分；此外，9%-16%比11%-17% SDS-PAGE凝胶对相对分子质量0-116 000(尤其是相对分子质量0-66 000)蛋白的分离更充分，且两者均极少损失小相对分子质量0-21 500的蛋白。 2.5 突触蛋白在双向电泳图中等电点和相对分子质量分布趋势从图3B-D中突触蛋白双向电泳图还可看出，突触蛋白在双向电泳中，等电点主要集中于pH 4-9区域，分子量大多数处于0-120 000，以10 000-70 000区域最集中。其后的MALDI-TOF/TOF质谱鉴定得到突触蛋白等电点和分子量信息也证实了这一结论。例如，Witzman等[7]进行了大鼠脑皮质突触全蛋白质组学分析(图3D)，用MALDI-TOF/ TOF质谱成功鉴定出163个突触蛋白点。实验对其成功鉴定突触蛋白的分子量和等电点信息进行了直方图分析，可以看出，突触蛋白等电点分布于pH 4-11区域(主要集中于pH 4-9，图4A)；相对分子质量分布于0-90 000(集中于10 000-70 000的范围，图4B)。又如，根据本课题组前期所做的慢性轻度应激抑郁大鼠与正常大鼠海马突触差异蛋白质组学实验数据[4, 27]，实验描绘了差异表达的突触蛋白的等电点和相对分子质量散点分布图，可以看出差异突触蛋白等电点分布于 pH 4-9区域内，相对分子质量处于10 000-110 000范围，以 20 000-70 000为其分布高峰(图4C)。"

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