同位素标签技术在人肾小管上皮细胞蛋白质组学研究中的应用

Application of isobaric tags labeling in proteomic research of human renal proximal tubular epithelial cells

目的建立并优化人肾小管上皮细胞蛋白质组学分析所需的相对和绝对定量的同位素标签试剂(iTRAQ)标记技术,为进一步研究奠定基础。方法将人肾小管上皮细胞株HK-2通过细胞裂解、丙酮沉淀、蛋白变性、还原及酶解,最后进行iTRAQ标记和质谱鉴定,研究iTRAQ在HK-2细胞的标记情况和蛋白质组学。结果通过质谱检测,检测到带有iTRAQ标记肽段。随机选取20个前体离子,通过3次重复试验,鉴定出iTRAQ标记多肽肽段分别为17、15和18个,标记效率在75.5%～90.0%,且组间标记效率差异无统计学意义(P>0.05)。结论iTRAQ可以在HK-2细胞中标记效果满意,且重复性较好,为进一步研究肾脏疾病的蛋白质组学奠定了基础。

Objective 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 renal proximal tubular epithelial cells. Methods After cell lysis, acetone precipitation, denaturation and protein digestion of human renal proximal tubular epithelial cell line HK-2, the peptide was labeled with iTRAQ reagents and subjected to mass spectrometry. The labeling of iTRAQ in HK-2 cells and proteomics were studied. Results Peptide tagged with iTRAQ was detected by mass spectrometry. Twenty precursors were randomly selected, and 17, 15 and 18 peptides tagged with iTRAQ were respectively detected for 3 repeated tests. The labeling effieiencies ranged between 75.5% and 90.0%, with no significant difference among groups （P 〉0. 05）. Conclusion The labeling of iTRAQ in HK-2 cells is successful with favourable reproducibility, which lays a foundation for the further research of proteomics in renal diseases.