[1]田晓琳,张赛航,孔德志,等.乳鼠及成年大鼠脑皮质膜蛋白差异的分析[J].中国药理学通报,2018,(08):1179-1184.[doi:10.3969/j.issn.1001-1978.2018.08.028]
 TIAN Xiao-lin,ZHANG Sai-hang,KONG De-zhi,et al.Analysis of difference of cerebral cortex membrane proteins between neonatal and adult rats[J].Chinese Pharmacological Bulletin,2018,(08):1179-1184.[doi:10.3969/j.issn.1001-1978.2018.08.028]
点击复制

乳鼠及成年大鼠脑皮质膜蛋白差异的分析()
分享到:

《中国药理学通报》[ISSN:/CN:]

卷:
期数:
2018年08期
页码:
1179-1184
栏目:
实验方法学
出版日期:
2018-07-26

文章信息/Info

Title:
Analysis of difference of cerebral cortex membrane proteins between neonatal and adult rats
文章编号:
1001-1978(2018)08-1179-06
作者:
田晓琳张赛航孔德志任雷鸣
河北医科大学中西医结合学院,河北 石家庄 050017
Author(s):
TIAN Xiao-linZHANG Sai-hangKONG De-zhiREN Lei-ming
College of Chinese Integrative Medicine,Hebei Medical University,Shijiazhuang 050017,China
关键词:
脑皮质组织 膜蛋白 跨膜蛋白提取 Orbitrap Fusion质谱仪 大鼠 乳鼠
Keywords:
cerebral cortex tissue membrane proteins transmembrane protein extraction Orbitrap Fusion mass spectrometer rats neonatal rats
分类号:
R-332; R322.81; R329.24; R341; R977.6
DOI:
10.3969/j.issn.1001-1978.2018.08.028
文献标志码:
A
摘要:
目的 建立基于Novagen ProteoExtract提取、NanoLC-MS/MS鉴定Sprague-Dawley大鼠脑皮层膜蛋白的组学方法,并用本法研究大鼠乳鼠及成年鼠皮层的差异膜蛋白。方法 分离大鼠皮层后,通过Novagen ProteoExtract 跨膜蛋白抽提试剂盒(简称TM-PEK)中的不同提取液(提取缓冲液2A与提取缓冲液2B)抽提蛋白,经Filter Aided Proteome Preparation(FASP)酶解、NanoLC 分离、Orbitrap Fusion高分辨质谱采集数据,通过Proteome Discover软件解析所提取到的蛋白,并对这些蛋白的疏水性、跨膜情况及生物信息学进行分析。结果 提取缓冲液2B可以鉴定出疏水性蛋白(GRAVY值>0)618个,具有跨膜区域(TMDs >1)的蛋白304个,而缓冲液2A仅能鉴定出48个疏水性蛋白和21个具有跨膜区域的蛋白,且Gene Ontology分析显示,提取液2B所抽提到的蛋白在膜(membrane)、膜部分(membrane part)和拟核(nucleoid)中明显富集。从新生大鼠和成年大鼠皮质的不同皮质蛋白中可以清楚地看到,大量涉及细胞间相互作用的突触蛋白在成年大鼠中明显富集,而新生大鼠中涉及蛋白质合成的蛋白明显高于成年鼠。结论 本研究建立了基于TM-PEK试剂盒提取、NanoLC-MS/MS分析鉴定大鼠皮层膜蛋白的方法,并研究了大鼠乳鼠及成年鼠皮层的差异膜蛋白,成年鼠以突触为代表的细胞间的联系发生了明显变化,而乳鼠蛋白的合成较成年鼠旺盛。
Abstract:
Aim To establish a membrane proteomics method using Novagen ProteoExtract extraction and NanoLC-MS/MS technologies,and to analyze the difference of membrane proteins from cerebral cortex tissue between neonatal and adult rats.Methods The rat cerebral cortical membrane proteins were extracted by two extract buffers(extraction buffer 2A and extraction buffer 2B)in the Novagen ProteoExtract Transmembrane Protein Extraction Kit(TM-PEK).After enzymatic hydrolysis by the filter aided proteome preparation(FASP)and membrane protein analysis by nano-liquid chromatography coupled with Orbitrap Fusion mass spectrometer,the proteins were identified by Proteome Discover software.The hydrophobicity,transmembrane status and bioinformatics of identified proteins were also analyzed.Results There were 618 hydrophobic proteins(GRAVY value greater than 0)identified by extraction buffer 2B(E2B),and 304/618 proteins had more than one predicted TMDs.There were only 48 hydrophobic proteins identified by extraction buffer 2A,and 21/48 proteins had more than one predicted TMDs.Gene Ontology analysis showed that the proteins extracted by E2B were significantly enriched in membrane,membrane part and nucleoid.A large amount of synaptic proteins referred to the intercellular interactions were significantly enriched in adult rat,whereas the proteins referred to protein synthesis were much more in neonatal rats than those in adult rats.Conclusions A method based on TM-PEK kit extraction and NanoLC-MS/MS technology has been established to analyze the rat membrane proteins from cerebral cortex tissue.The proteins related to synapses that connect neurons in the brain are much more in adult rats when compared with neonatal ones,whereas the protein synthesis ability is weaker in neonatal rats.

参考文献/References:

[1] 曹莉莎,张 芳,罗 文,等.抑郁大鼠海马组织的比较蛋白质组学研究[J].中国药理学通报,2016,32(5):697-702. [1] Cao L S,Zhang F,Luo W,et al.Analysis of CUMS-induced differentially expressed proteins in rat hippocampus by ITRAQ [J].Chin Pharmacol Bull,2016,32(5):697-702.
[2] Boyd R S,Jukes-Jones R,Walewska R,et al.Protein profiling of plasma membranes defines aberrant signaling pathways in mantle cell lymphoma[J].Mol Cell Proteomics,2009,8(7):1501-15.
[3] Heo S,Spoerk S,Birner-Gruenberger R,et al.Gel-based mass spectrometric analysis of hippocampal transmembrane proteins using high resolution LTQ Orbitrap Velos Pro [J].Proteomics,2014,14(17-18):2084-8.
[4] 刘婧媛,武丽南,杨元辉,等.LC-MS/MS法测定家兔血浆中卤米松浓度及其药动学研究[J].中国药理学通报,2017,33(3):411-6.
[4] Liu J Y,Wu L N,Yang Y H,et al.Determination of halometasone and its pharmacokinetics study in rabbit plasma by LC-MS/MS [J].Chin Pharmacol Bull,2017,33(3):411-6.
[5] Erde J,Loo R R,Loo J A.Enhanced FASP(eFASP)to increase proteome coverage and sample recovery for quantitative proteomic experiments[J].J Proteome Res,2014,13(4):1885-95.
[6] Huang da W,Sherman B T,Lempicki R A.Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources [J].Nat Protoc,2009,4(1):44-57.
[7] Lin Y,Liu H,Liu Z,et al.Shotgun analysis of membrane proteomes using a novel combinative strategy of solution-based sample preparation coupled with liquid chromatography-tandem mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci,2012,901:18-24.
[8] Chen P,Li X,Sun Y,et al.Proteomic analysis of rat hippocampal plasma membrane:characterization of potential neuronal-specific plasma membrane proteins [J].J Neurochem,2006,98(4):1126-40.
[9] Savas J N,Stein B D,Wu C C,et al.Mass spectrometry accelerates membrane protein analysis [J].Trends Biochemical Sci,2011,36(7):388-96.
[10] Alfonso-Garrido J,Garcia-Calvo E,Luque-Garcia J L.Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry [J].Anal Bioanal Chem,2015,407(17):4893-905.
[11] Kang S U,Heo S,Lubec G.Mass spectrometric analysis of GABAA receptor subtypes and phosphorylations from mouse hippocampus [J].Proteomics,2011,11(11):2171-81.
[12] 刘 娟,朱建航,范杰平,等.去污剂/聚合物双水相体系在膜蛋白分离中的应用[J].江西科学,2008,26(3):416-20.
[12] Liu J,Zhu J H,Fang J P,et al.Application of detergent/polymer aqueous two phase systems for purification of membrane protein[J].Jiangxi Sci,2008,26(3):416-20.
[13] 柯冬蕾,李和平,徐境羚,等.马鹿精子膜蛋白提取及理化性质分析[J].东北林业大学学报,2008,36(11):83-5.
[13] Ke D L,Li H P,Xu J L,et al.Extraction of sperm membrane proteins from cervus elaphus and its physicochemical properties[J].J Northeast For Univ,2008,36(11):83-5.

备注/Memo

备注/Memo:
收稿日期:2018-03-19,修回日期:2018-04-18
基金项目:河北省自然科学基金资助项目(No H2016206030); 河北医科大学科研发展基金(No kyfz 064)
作者简介:田晓琳(1992-),女,硕士生,研究方向:蛋白质组学,E-mail:809401215@qq.com;
任雷鸣(1956-),男,博士,教授,博士生导师,研究方向:心血管及自主神经药理学,通讯作者,E-mail:ren-leiming@263.net
更新日期/Last Update: 2018-07-26