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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。
The establishment and optimization of the relative and absolute quantitative isotopic tag (iTRAQ) multiplex labeling techniques required for proteomic analysis of human peripheral blood mononuclear cells lay the foundation for further research. Human peripheral blood mononuclear cells (hPBMCs) were isolated and the total cellular protein was extracted. After digestion with trypsin, iTRAQ labeling and tandem mass spectrometry were used. Three replicate experiments were carried out to select 30 precursor ions each time for mass spectrometry. The number of peptides with iTRAQ labeled reporter groups was 26, 28 and 29, respectively. The labeling efficiency was 86.7% ~ 96.7% There was no significant difference between the two groups (P> 0.05). The coefficient of variation (CV) of the relative quantification ratios of different proteins was 7.6% ~ 25.5%, with no significant difference between the two groups (P> 0.05) The relative coefficient of variation of the coefficient of variation was 9.3% ~ 19.1%, and there was no significant difference between the two groups (P> 0.05). The results showed that iTRAQ multiplex labeled tandem mass spectrometry could be used to label hPBMC protein. The labeling efficiency was high, the repeatability and accuracy were good, which laid the foundation for the further study of quantitative proteomics of PBMC in a variety of autoimmune diseases.