CAJ | 학술논문

在磷酸化蛋白质组学研究中,根据是否需要对待富集样品进行甲酯化处理,可将固化金属离子亲和层析(IMAC)方法分为两类,即需要甲酯化处理的IMAC方法(ME-IMAC)和不需要甲酯化处理的IMAC方法(Non-MEIMAC).要实现对磷酸化多肽的有效富集和鉴定,就必须对富集方法进行选择和优化.利用基质辅助激光解析离子化串联飞行时间质谱(MALDI-TOF-MS)对两种方法富集的磷酸化多肽进行了比较研究.结果表明,ME-IMAC方法容易发生样品丢失,质谱结果的分析也比较复杂,而Non-ME-IMAC方法则不仅操作简单而且富集效果理想.另外,优化了Non-ME-IMAC方法的实验条件,指出最佳的结合溶液是8%ACN/0.3%TFA,最佳的洗脱溶液是0.1mol/L EDTA(pH值8.0),从而建立了一套完整而简单有效的磷酸化多肽富集方法.
재린산화단백질조학연구중,근거시부수요대대부집양품진행갑지화처리,가장고화금속리자친화층석(IMAC)방법분위량류,즉수요갑지화처리적IMAC방법(ME-IMAC)화불수요갑지화처리적IMAC방법(Non-MEIMAC).요실현대린산화다태적유효부집화감정,취필수대부집방법진행선택화우화.이용기질보조격광해석리자화천련비행시간질보(MALDI-TOF-MS)대량충방법부집적린산화다태진행료비교연구.결과표명,ME-IMAC방법용역발생양품주실,질보결과적분석야비교복잡,이Non-ME-IMAC방법칙불부조작간단이차부집효과이상.령외,우화료Non-ME-IMAC방법적실험조건,지출최가적결합용액시8%ACN/0.3%TFA,최가적세탈용액시0.1mol/L EDTA(pH치8.0),종이건립료일투완정이간단유효적린산화다태부집방법.
In phosphoproteomies,according to whether the sample treated with methyl esterifieation(ME)necessarily or not,immobilized-metal ion affinity chromatography(IMAC)can be divided into two kinds,ME-IMAC and Non-ME-IMAC.In order to find out better one of them.they were compared by MALDI-TOF-MS.The results firstly showed that ME-IMAC method could cause adsorptive losses easily and complieme the MS analysis and data interpretation:in contrast,Non-ME-IMAC method was a simpler and more efficient strategy for phosphopeptide enrichment.In addition,the optimization of experimental conditions of Non-ME-IMAC showed that the best binding buffer was 8% ACN/0.3% IFA and the best eluting buffer was 0.1 mol/L EDTA(pH 8.0).The Non-ME-IMAC method reported here is a good reference to more and more researchers in phosphoproteomics.