摘要
建立并优化人外周血单个核细胞蛋白质组学分析所需的相对和绝对定量的等量异位标签(iTRAQ)多重标记技术,为进一步研究奠定基础。分离人外周血单个核细胞(hPBMC),抽提细胞总蛋白,经胰酶消化后进行iTRAQ标记和串联质谱检测。通过3次重复试验,每次随机选取30个前体离子进行质谱检测,检测到带有iTRAQ标记报告基团的肽段分别为26、28和29个,标记效率为86.7%～96.7%,且组间差异无统计学意义(P>0.05);不同蛋白的肽段相对定量比值的变异系数为7.6%～25.5%,且组间差异无统计学意义(P>0.05);同一蛋白的不同肽段相对定量比值的变异系数为9.3%～19.1%,且组间差异无统计学意义(P>0.05)。结果表明iTRAQ多重标记串联质谱技术可用于hPBMC蛋白的标记,且标记效率高,重复性和准确性好,为进一步研究多种自身免疫性疾病患者的PBMC定量蛋白质组学奠定了基础。
This paper is aimed to establish and optimize proteomic research platform using isobaric tags for relative and absolute quantitation（iTRAQ） so as to facilitate further proteomic research of human peripheral blood mononuclear cells（hPBMC）.We collected hPBMC,after protein extraction and trypsin digestion,we labeled the samples with iTRAQ reagents and then subjected to mass spectrometry.In triplicates,thirty precursors were randomly selected and detected;as a result,26,28 and 29 peptides were respectively tagged with iTRAQ reporter ions.The labeling efficiencies ranged between 86.7%-96.7%,with no significant difference among the groups（P0.05）.The coefficient of variance for the relative ratios of peptides from different proteins was ranged from 7.6% to 25.5% and there were no significant differences across the groups（P0.05）.The coefficient of variance for the relative ratios of different peptides from the same protein was varied from 9.3% to 19.1% and the differences across groups were not significant（P0.05）.The labeling of iTRAQ combined with tandem mass spectrometry in PBMC was successful with favourable reproducibility and accuracy,which could lay a foundation for further proteomic study of hPBMC in autoimmune disorders.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
2011年第3期538-542,共5页
Journal of Biomedical Engineering
基金
深圳市科技计划重点项目资助(201001001)
关键词
相对和绝对定量的等量异位标签
人外周血单个核细胞
蛋白质组学
质谱
Isobaric tags for relative and absolute quantitation（iTRAQ）
Human peripheral blood mononuclear cells（hPBMC）
Proteomics
Mass spectrometry
