iTRAQ试剂结合二维液相色谱质谱联用技术对蛋白质进行鉴定和比较定量研究（英文）  

Identification and comparative quantification of proteins using iTRAQ reagent combined with two-dimensional liquid chromatography and mass spectrometry

作　　者：胡炜[1,2] 付强[1,2] 周洁[1,2] 

机构地区：[1]广西大学亚热带农业生物资源保护与利用国家重点实验室 [2]广西大学生命科学与技术学院,广西南宁530004

出　　处：《广东农业科学》2014年第15期93-99,110,共8页Guangdong Agricultural Sciences

基　　金：Scientific Research Fund of Guangxi Education Department(YB2014004);The Open topic from Guangxi Key Laboratory of Buffalo Genetics Reproduction and Breeding(SNKF-2012-05,SNKF-2013-02)

摘　　要：采用相对和绝对定量同位素标签(iTRAQ)对蛋白质进行鉴定和比较定量,其能进一步比较更为复杂样品的蛋白质表达水平。将蛋白质样品用胰蛋白酶酶解,分别被几种分子量相等的iTRAQ标签中的一种标记,然后混合。混合后将被标记的多肽通过强阳离子交换色谱(SCX)和纳升反相色谱(RP)分离,结合质谱(MS)分析。结果表明,在保证95%的置信度下,共有54个唯一肽段和6个标准蛋白质被成功鉴定,包括大肠杆菌的β-半乳糖苷酶、β-乳球蛋白、小鸡溶菌酶,人血清铁传递蛋白的蛋白质定量表达水平在样品A和样品B中未表现出显著差异,但样品B中牛血清白蛋白(BSA)和α-乳白蛋白的数量均为样品A的一半。该结果与预期试验结果相符,且重复性好,表明此方法具有稳定性和可重复性,适用于蛋白质鉴定和表达水平的比较定量研究,此外,其能显著提高蛋白质混合物的研究通量。Identification and comparative quantification between protein samples were performed using isobaric tags for relative and absolute quantification（iTRAQ）,which could move forward to compare protein expression levels among more complex samples.Protein samples subjected to enzymatic digestion of trypsin were labeled with one of several chemically identical iTRAQ tags respectively before pooled,and then the mixed peptides labeled were separated and analyzed via strong cation exchange（SCX） chromatography followed by nano reverse phase（RP） chromatography combined with mass spectrometry（MS）.A total of 54 unique peptides and 6 standard proteins were identified successfully with 95% confidence,including Escherichia coli β-galactosidase,β-lactoglobulin,chick lysozyme.Human serotransferrin in sample A did not show significant difference at protein quantitative expression levels compared with that in sample B,whereas the quantity of bovine serum albumin（BSA） and α-lactalbumin in sample B cut down to half of that in sample A.The results correspond to the designed experiments,and the repeatability test shows that the approach is stable and reproducible.It reveals the technique is well suitable for the identification and comparative quantitative studies of protein expression levels,in addition,which can significantly increase the investigative throughput of protein mixtures.

关 键 词：ITRAQ 比较定量 蛋白质表达水平 

分 类 号：S96[农业科学—水产养殖]