技术服务当前位置:首页技术服务 >

SDS-PAGE凝胶电泳结合LTQ质谱分析

来源:奔驰糖果派对攻略 作者:生物质谱实验室 时间:2009-04-21 点击:

应用实例: 

reference
Wantao Ying , Yangjun Zhang,Songfeng, Jinglan Wang, Yun Cai, Xiaohong Qian,et.al.
A Dataset of Human Fetal Liver Proteome Identified by Subcellular Fractionation and
Multiple Protein Separation and IdentificationTechnology. Molecular & Cellular Proteomics 2006,5,1703-1707

Introduction
By doing the analysiswith integrated protein, expressed sequence tag, and genome datasets, 223 proteins and 15 peptides were complementarily identified with high quality MS/MS data.

Methods
1. TECHNOLOGY FOR PROTEIN EXPRESSION PROFILE SDS-PAGE Separation—Proteins were separated by SDS-PAGEwith different cross-linking percentages, 15, 10, and7.5%, to obtain a full representation of proteins ranging from 5 kDa to more than 300 kDa. After separation, these gels were stained with Colloidal Coomassie Blue R250, and the gel lanes were manually excised from loading position to the bottom of the gel. After in-gel digestion with trypsin, the extracted peptide mixtures were loaded onto nanoscale LCESI-Q-TOF MS or micro-LC-ion trap MS systems for protein identifications.
2. DATABASE QUERIES AND PROTEIN IDENTIFICATIONS
When the data produced by ESI-Q-TOF MS was searched against the IPI_human_2.33 protein database by MASCOT,mass tolerance of peptide precursor and its daughter ions
was set at 0.2 Da in peptide sequence tag, and one possible missed cleavage for trypsin digestion was selected. Protein identifications were performed based on probability-based
Mowse scoring algorithm with a confidence level of 95%.
3. PROTEIN EXPRESSION PROFILE OF HFL
4. COMPLEMENTARY IDENTIFICATION AND NOVEL PROTEIN MINING