CAJ | 학술논문

建立了基于在线二维弱阴离子交换色谱-反相液相色谱(2D-WAX-RPLC)的蛋白质分离系统,并用于不同生长时期鹿茸的比较蛋白质组分析.以5种标准蛋白质的混合物为样品,考察了系统的重现性.通过改变标准蛋白质之间的浓度比,研究了该系统进行蛋白质相对定量的能力.在此基础上,针对4个不同生长时期的鹿茸样品,分别采用5种不同的方法进行蛋白质提取,经2D-WAX-RPLC分离后,收集各生长时期对应蛋白质的峰高最大比超过2的组分.酶解后,采用微柱反相液相色谱-串联质谱(μRPLC-MS/MS)进行肽段的分离鉴定;共从9个差异蛋白质峰中鉴定到了22个差异蛋白质.研究结果表明,利用基于蛋白质水平的在线二维液相色谱分离技术找寻差异蛋白质,具有重现性好、自动化程度高等优点,可用于开展比较蛋白质组学的研究.
건립료기우재선이유약음리자교환색보-반상액상색보(2D-WAX-RPLC)적단백질분리계통,병용우불동생장시기록용적비교단백질조분석.이5충표준단백질적혼합물위양품,고찰료계통적중현성.통과개변표준단백질지간적농도비,연구료해계통진행단백질상대정량적능력.재차기출상,침대4개불동생장시기적록용양품,분별채용5충불동적방법진행단백질제취,경2D-WAX-RPLC분리후,수집각생장시기대응단백질적봉고최대비초과2적조분.매해후,채용미주반상액상색보-천련질보(μRPLC-MS/MS)진행태단적분리감정;공종9개차이단백질봉중감정도료22개차이단백질.연구결과표명,이용기우단백질수평적재선이유액상색보분리기술조심차이단백질,구유중현성호、자동화정도고등우점,가용우개전비교단백질조학적연구.
A method of on-line two-dimensional weak anion exchange chromatography-reversed-phase liquid chromatography (2D WAX-RPLC) was established,and applied for the comparative proteome study of antlers with four different growing stages.A five-protein-mixture was used as the sample to evaluate the reproducibility of the system.In addition,the capacity of such a system for the relative quantitative analysis was demonstrated by analyzing protein mixtures with different concentration ratios.Furthermore,five different lysis buffers were used to extract proteins from antlers with different growing stages.After the protein separation with 2D WAX-RPLC,the fractions with maximum peak height ratio larger than 2 were collected,digested,and further identified by micro-reversed-phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS).In total,22 differential proteins were identified from 9 fractions.Our experimental results demonstrated that 2D WAX-RPLC based protein separation has the advantages of good reproducibility and automation,and might be a complementary method of two-dimensional gel electrophoresis for comparative proteome study.