摘要
在使用对二甲基亚砜(DMSO)和NEDD8激活酶抑制剂(MLN4924,MLN)刺激的人脐静脉内皮细胞(HUVEC)内的蛋白质进行分析的过程中,利用Progenesis LC-M S软件对色谱图进行了保留时间的校正,并比较了同组分多次重复实验中的谱图相似率及两种刺激下细胞内蛋白质色谱图的相似度。样品经双酶切处理后,加入QconC AT标准蛋白质混合物作为参照,经高效液相色谱-串级质谱分离,后续又对谱图进行了校正与分析。经过谱图校正,将蛋白质鉴定结果从7 000个左右提高到8 000个以上,提高了蛋白质的鉴定效率。在利用谱图计数进行相对定量时,还分析了DMSO和MLN分别刺激HUVEC后细胞内的蛋白质差异在1 000个左右,并给出了校正后的色谱总离子流图的相似度比较。相比其他方法更为简单快捷和流程化,具有高通量高灵敏度的优点。
In the analysis of proteins in human umbilical vein endothelial cells（ HUVEC）trea-ted with dimethyl sulfoxide （ DMSO ） and NEDD8-activating enzyme inhibitor （ MLN4924, MLN）,the Progenesis LC-MS software（ Nonlinear Dynamics Ltd）was applied to liquid chro-matography spectrum alignment,while spectrum similarities were figured out among several experiments of the same sample,and also among different samples. After double enzymolysis, the sample was added with digested QconCAT standard proteins. They were separated by HPLC-MS / MS,followed by spectrum alignment and data analysis. This established experiment flow offered a better identification result of more than 8 000 proteins,while the original result was about 7 000 proteins,ensuring a relatively high identification efficiency. On the basis of rel-ative quantification with spectrum count,the described procedure can analyze the differential expression of proteins induced by DMSO and MLN. The similarities of total ion chromatograms after alignment were also compared. This method was proved to be quick and easy,with the advantages of high throughput and high sensitivity.
出处
《色谱》
CAS
CSCD
北大核心
2014年第4期349-354,共6页
Chinese Journal of Chromatography
基金
国家重大科学计划项目(2010CB912700
2013CB911201
2011CB910600)
国家高技术研究发展计划项目(2012AA020203
2014AAQ00361)
国家自然科学基金重点项目(21227805)
关键词
高效液相色谱-串级质谱
谱图校正
蛋白质分析
high performance liquid chromatography-tandem mass spectrometry（ HPLC-MS /MS）
spectrum alignment
protein analysis
