Statistical Analysis of the Experimental Variation in the Proteomic Characterization of Human Plasma by Two-Dimensional Difference Gel Electrophoresis

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• 作者：Todd H. Corzett ; Imola K. Fodor ; Megan W. Choi ; Vicki L. Walsworth ; Brett A. Chromy ; Kenneth W. Turteltaub ; Sandra L. McCutchen-Maloney
• 刊名：Journal of Proteome Research
• 出版年：2006
• 出版时间：October 2006
• 年：2006
• 卷：5
• 期：10
• 页码：2611 - 2619
• 全文大小：472K
• 年卷期：v.5,no.10(October 2006)
• ISSN：1535-3907

文摘

The complexity of human plasma presents a number of challenges to the efficient and reproducibleproteomic analysis of differential expression in response to disease. Before individual variation anddisease-specific protein biomarkers can be identified from human plasma, the experimental variabilityinherent in the protein separation and detection techniques must be quantified. We report on thevariation found in two-dimensional difference gel electrophoresis (2-D DIGE) analysis of human plasma.Eight aliquots of a human plasma sample were subjected to top-6 highest abundant protein depletionand were subsequently analyzed in triplicate for a total of 24 DIGE samples on 12 gels. Spot-wisestandard deviation estimates indicated that fold changes greater than 2 can be detected with amanageable number of replicates in simple ANOVA experiments with human plasma. Mixed-effectsstatistical modeling quantified the effect of the dyes, and segregated the spot-wise variance intocomponents of sample preparation, gel-to-gel differences, and random error. The gel-to-gel componentwas found to be the largest source of variation, followed by the sample preparation step. An improvedprotocol for the depletion of the top-6 high-abundance proteins is suggested, which, along with theuse of statistical modeling and future improvements in gel quality and image processing, can furtherreduce the variation and increase the efficiency of 2-D DIGE proteomic analysis of human plasma.