基于磁珠处理MALDITOF实验技术优化.docVIP

基于磁珠处理MALDITOF实验技术优化 　【摘要】 目的：利用基质辅助激光解析离子化飞行时间质谱（matrixassisted laser desorption/ionization timeofflight mass spectrometry，MALDITOF MS）技术检测血清中蛋白，目前常用磁珠来分离提纯，本研究目的是寻找一套有效的血清样品收集、处理、存储方法，应用于蛋白组学分析。方法：采用磁珠分离提纯人体血清，利用MALDITOF技术检测1 000～10 000 Da范围蛋白或多肽，考察其重复性及血清不同的冻融次数和基质与样品搭配比例对结果的影响。结果：人类血清的质谱图在1 000～10 000 Da范围内可检测到230多个峰，重复性极好。样品多次冻融对小分子量的多肽有一定影响。在适当范围内改变样品与基质比例对结果无影响。结论：基于磁珠分离提纯的MALDITOF技术在应用于人体血清检测时应用标准化程序可以减少变异。 【关键词】 蛋白质组学 磁珠疗法 基质辅助 激光解析离子化飞行时间质谱 【ABSTRACT】 Objective:Magnetic bead purification for the analysis of proteins in human blood serum facilitates the identification of potential new biomarkers with matrixassisted laser desorption/ionization timeofflight mass spectrometry（MALDITOF MS).The purpose of the study was to establish a proteome fractionation technique and to validate a standardized blood sampling,processing,and storage procedure for proteomic pattern analysis.Methods:Magnetic bead separation was used for proteome profiling of human blood by MALDITOF MS（mass range,1 000～10 000 Da)and the effects on the reproducibility of the proteome analysis of the number of freezethaw cycles of samples and ratio of sample and matrix were studied.Results:The proteome pattern of human serum was characterized by 230 signals in the mass range of 1 000～10 000 Da.Serum mass patterns differed between samples in different freezethaw cycles,espeaially in lowweight molecule peptides.Different ratios of sample and matrix showed no effects on proteome profiling.Conclusion:Application of the standardized preanalytical blood sampling and storage procedure in combination with magnetic beadbased fraction decreases variability of proteome patterns in human serum assessed by MALDITOF MS. 【KEY WORDS】 Proteomics,Magnetic Bead Therapy,MALDITOF MS 利用质谱进行蛋白质组学研究是近年兴起的、较为有效的研究方法，特别对于发现潜在的生物标志物，用以鉴别健康与疾病具有重大意义[1，2]。SELDITOF和MALDITOF技术在用于发现生物标志物方面国内外均有报道，他们与以往的双向电泳、多维液相色谱技术不同，灵敏度更高，而且工作量减少，节省时间。SELDITOF主要是将待测样品的多肽或蛋白片断浓缩于经过修饰的靶面，直接上质谱。其最早的临床报道是鉴别癌症，例如卵巢癌、前列腺癌、膀胱癌、乳腺