摘要
目的建立基于Novagen Proteo Extract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 Proteo Extract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、Nano LC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TMPEK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。
Aim To establish a membrane proteomics method using Novagen Proteo Extract 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 2 A and extraction buffer 2 B） in the Novagen Proteo Extract 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 2 B（ E2 B）,and 304/618 proteins had more than one predicted TMDs. There were only 48 hydrophobic proteins identified by extraction buffer2 A,and 21/48 proteins had more than one predicted TMDs.Gene Ontology analysis showed that the proteins extracted by E2 B 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 Nano LC-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.
作者
田晓琳
张赛航
孔德志
任雷鸣
TIAN Xiao-lin;ZHANG Sai-hang;KONG De-zhi;REN Lei-ming(College of Chinese Integrative Medicine,Hebei Medical University,Shijiazhuang 050017,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2018年第8期1179-1184,共6页
Chinese Pharmacological Bulletin
基金
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)
