A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction:apolipoprotein H(APOH),apolipoprotein J(APOJ),apolipoprotein A4(APOA4),apolipoprotein E(APOE),and apolipoprotein D(APOD).The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600,followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500.Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20%higher than corresponding non-labeled peptides.Through optimized transitions and parameters,we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins.The results indicated that the MRM signals of four target APOs were significantly increased after depletion,while the MRM signal of one APO,APOD,was decreased.Furthermore,the relative abundances of the five target APOs in healthy human plasma were stable,and the ranking of these proteins according to their MS responses changed slightly.Therefore,we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.
LI WenWenWANG QuanHuiCHEN JianJunZHOU JianZHOU XinYuXIE Peng
目的：优化尿液蛋白质制备方法以获得蛋白点数多且分辨率高的尿液双向凝胶电泳（two dimensional gel electrophore—sis，2-DE）图谱，为尿液蛋白质组学研究提供技术支撑。方法：首先，使用6种不同方法制备尿液蛋白质后比较蛋白质得率，并分析这些方法的2一DE谱蛋白点数及3次重复匹配率，进行统计分析计算P值；最后利用2种固相pH梯度（immobilized pH gra—dient，IPG）胶条和不同的聚丙烯酰胺凝胶（polyacrylamide gel electrophoresis，PAGE）胶浓度进行2-DE分析，比较所得蛋白图谱的蛋白点数及分辨率。结果：与其他方法相比，超滤离心法制备尿液蛋白能获得更多的蛋白总量[（791±17）Ixg，P=0．000]，得到的电泳图谱蛋白点数也更多[（724±29）个，P=0．000]；利用pH3—10IPG胶条和12．5％PAGE分离有利于尿液蛋白质的整体分析，特别是酸性和碱性蛋白质点信息，而pH4—7IPG胶条能使等电点为4—7蛋白质的2-DE谱分辨率更高（P〈0．05）。结论：建立了与2-DE兼容的尿液蛋白质制备方法．获得了更为全面的尿液蛋白点信息，为临床生理和疾病的尿液蛋白质组学研究奠定了基础。
