iTRAQ技术在颅内动脉瘤病人血浆差异表达蛋白检测中的应用

目的探讨同位素标记相对和绝对定量(iTRAQ)技术在颅内动脉瘤血浆差异表达蛋白(DEPs)检测中的应用价值。方法收集2019年1~12月收治的10例颅内破裂动脉瘤病人血浆10份,同时收集年龄、性别、吸烟史、饮酒史、高血压以及动脉瘤位置等相匹配的10例颅内未破裂动脉瘤血浆10份作为对照组。采用iTRAQ技术筛选血浆样本DEPs,应用生信分析方法GO分析和KEGG通路富集分析,并与GEO数据库的数据集进行交叉验证。结果经蛋白质组学分析鉴定出806个可定量蛋白,其中DEPs共122个,表达上调59个,表达下调63个。GO和KEGG分析结果显示,动脉瘤破裂主要与炎症和免疫反应相关。DEPs相互作用网络分析显示,白蛋白、血红蛋白β亚基、钙调蛋白1、转铁蛋白和载脂蛋白A1是网络核心蛋白。与GEO数据集交叉结果显示,血浆样本SLMAP、IQGAP3、PTPRJ、PEBP1、S100P、BASP1和AOC3等表达水平与GEO数据集的动脉瘤组织基因表达水平呈现相同变化趋势,而且破裂动脉瘤与未破裂动脉瘤之间具有统计学差异(P<0.05)。结论本文结果初步筛选出与颅内动脉瘤破裂相关的血浆DEPs,这些DEPs与炎症和免疫反应密切相关。这为研究颅内动脉瘤发病的分子机制和潜在的治疗靶点提供了新的线索。

iTRAQ多重化学标记串联质谱技术在生命科学领域中的应用

同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术—利用多种同位素试剂标记蛋白多肽N末端或赖氨酸侧链基团,经高精度质谱仪串联分析,可同时比较多达8种样品之间的蛋白表达量,是近年来定量蛋白质组学常用的高通量筛选技术,正迅速被广大学者所接受。主要从iTRAQ实验技术概要、iTRAQ多重化学标记串联质谱技术在生命科学领域中的应用、问题与展望等三个方面进行简要总结。

Study on the Application of iTRAQ Technology in Retinal Apoptosis Protein in Diabetic Retinopathy Mice

Objective:Using isobaric tags for relative and absolute quantitation(i TRAQ)technology to study differential protein expression in the retinal tissue of DR mouse models,providing proteomic evidence at the protein level for the pathogenesis of DR.Methods:Firstly,establish diabetic mice and DR mice models,use i TRAQ technology to detect the retinal tissue samples of normal control group and DR model group mice,label the total retinal proteins of mice with i TRAQ reagent,and analyze them using mass spectrometry technology.Evaluate the differential proteins of the two groups using BioWorks TM 3.0 software,and conduct interaction feature analysis on the different proteins using the STRING website.Results:The i TRAQ technology detected a total of 406 different proteins between the diabetes and normal control groups,with 19 of them closely related to retinal cell apoptosis.Among them,significantly different proteins include acyl-CoA dehydrogenase short chain specific(ACADS),ataxin-10(ATXN10),BCL-2-associated X protein(BAX),caspase-3(CASP3),collagen type IIα1 chain(COL4A2),glycyl-tRNA synthetase(GARS),glial fibrillary acidic protein(GFAP),legumain(LGMN),mucin-4(MUC4),N-myc downstream-regulated gene 1 protein(NDRG1),with ratios to internal controls in the normal group of 1.67,2.06,1.76,2.16,1.53,1.87,1.24,1.61,0.42,0.56,respectively;and there is a potential functional association between GFAP,CASP3,and BAX proteins.Conclusion:In DR mice retinas,there are abnormal changes in the expression of a large number of apoptosis-related proteins.i TRAQ technology can effectively screen out key apoptosis proteins,among which GFAP,CASP3,and BAX may have adverse effects on the progression of DR by participating in the apoptosis process.The application of i TRAQ technology can provide new technical support for proteomic research on apoptosis proteins.

iTRAQ多重化学标记串联质谱技术在蛋白质组学中的应用

i TRAQTM是2004年美国应用生物系统公司(Applied Biosystems Incorporation,ABI)最新推出的一种多肽体外标记试剂,该技术可对复杂样本、细胞器、细胞裂解液等样本进行相对和绝对定量研究,具有较好的定量效果、较高的重复性,并且弥补了DIGE及ICAT的不足,正迅速被广大学者所接受。主要从i TRAQ实验原理、实验流程、i TRAQ与其他定量蛋白质组学方法的比较等3个方面进行简要总结。