TiO2 with Tandem Fractionation (TAFT): An Approach for Rapid, Deep, Reproducible, and High-Throughput Phosphoproteome Analysis,Journal of Proteome Research

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TiO2 with Tandem Fractionation (TAFT): An Approach for Rapid, Deep, Reproducible, and High-Throughput Phosphoproteome Analysis
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2017-11-15 00:00:00 , DOI: 10.1021/acs.jproteome.7b00520
Liangliang Ren _{1,

2} , Chaoying Li _{1,

2} , Wenli Shao _{1,

2,

3} , Weiran Lin _{1,

2} , Fuchu He _{1,

2} , Ying Jiang _{1,

2}

Affiliation

Mass-spectrometry-based phosphoproteomic workflows traditionally require efficient prefractionation and enrichment of phosphopeptides to gain an in-depth, global, and unbiased systematic investigation of phosphoproteome. Here we present TiO₂ with tandem fractionation (TAFT) approach, which combines titanium dioxide (TiO₂) enrichment and tandem high-pH reverse-phase (HpRP) for phosphoproteome analysis in a high-throughput manner; the entire workflow takes only 3 h to complete without laborious phosphopeptide preparation. We applied this approach to HeLa and HepG2.2.15 cells to characterize the capability of TAFT approach, which enables deep identification and quantification of more than 14 000 unique phosphopeptides in a single sample from 1 mg of protein as starting materials in <4 h of MS measurement. In total, we identified and quantified 21 281 phosphosites in two cell lines with >91% selectivity and high quantitative reproducibility (average Pearson correlation is 0.90 between biological replicates). More generally, the presented approach enables rapid, deep, and reproducible phosphoproteome analysis in a high-throughput manner with low cost, which should facilitate our understanding of signaling networks in a wide range of biological systems or the process of clinical applications.

中文翻译：

串联分离（TAFT）的TiO ₂：一种快速，深层，可重现和高通量磷酸化蛋白质组分析的方法

传统上，基于质谱的磷酸化蛋白质组学工作流程需要进行有效的预分离和磷酸肽富集，以获得对磷酸化蛋白质组学的深入，全局且无偏见的系统研究。在这里，我们本的TiO ₂与串联分馏（TAFT）的方法，它结合了二氧化钛（TiO ₂）富集和串联高pH反相（HpRP）以高通量方式进行磷酸化蛋白质组分析; 无需费力的磷酸肽制备，整个工作流程仅需3小时即可完成。我们将这种方法应用于HeLa和HepG2.2.15细胞，以表征TAFT方法的功能，该方法能够在不到4小时的MS中从1 mg蛋白质作为起始材料对单一样品中的14 000多种独特磷酸肽进行深度鉴定和定量。测量。总的来说，我们鉴定和定量了两个细胞系中的21 281个磷酸位点，具有> 91％的选择性和较高的定量再现性（生物学重复之间的平均Pearson相关性为0.90）。更一般而言，本文提出的方法可实现高通量，低成本，快速，深入和可重现的磷酸化蛋白质组分析，

更新日期：2017-11-16

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