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Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows
Journal of Proteome Research ( IF 4.4 ) Pub Date : 2018-02-13 00:00:00 , DOI: 10.1021/acs.jproteome.7b00840
Ekaterina Stepanova 1 , Steven P. Gygi 1 , Joao A. Paulo 1
Affiliation  

High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform–methanol (C–M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (n = 3), C–M (n = 2), and FPD using either 10 kDa (n = 3) or 30 kDa (n = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows.

中文翻译:

基于过滤器的蛋白质消化(FPD):TMT工作流程的无洗涤剂和基于支架的策略

高通量蛋白质组图谱分析需要彻底优化以实现全面分析。我们开发了一种类似于过滤辅助的样品制备(FASP)的无洗涤剂方法,称为基于过滤的蛋白质消化(FPD)。我们将FPD与基于等压标记的蛋白质组分析中常用的蛋白质提取方法(即三氯乙酸(TCA)和氯仿-甲醇(CM)沉淀)进行了比较。我们从SH-SY5Y神经母细胞瘤细胞系中分离出哺乳动物全细胞裂解液,用10 kDa(n = 3)或30 kDa的TCA(n = 3),CM(n = 2)和FPD进行平行蛋白质加工(n= 3)分子量截止膜。我们用串联质量标签(TMT)试剂标记每个样品,以构建TMT11-plex实验。总共在所有样品中定量了8654个蛋白质。配对比较显示两种FPD方法之间单个蛋白质丰度测量值的偏差很小,而TCA和FPD显示的差异最大。具体而言,当使用TCA沉淀处理样品时,与其他测试方法相比,膜蛋白更容易定量。但是,在全球范围内,在最不同的一对蛋白质提取方法(即FPD10和TCA)中,只有4%的蛋白质差异大于4倍。我们得出的结论是,无洗涤剂的FPD策略(特别是使用流动速度更快的30 kDa过滤器)是对高通量TMT工作流程的无缝改变。
更新日期:2018-02-14
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