Journal
JOURNAL OF PROTEOME RESEARCH
Volume 17, Issue 1, Pages 710-721Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.7b00520
Keywords
TAFT; HpRP; phosphopeptide; phosphoproteome; titanium dioxide; enrichment; chromatography; fractionation; label-free; quantification
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Funding
- Chinese State Key Projects for Basic Research (973 Program) [2014CBA02001, 2013CB910502]
- National Key Research and Development Project [2016YFC0902400, 2017YFC0906603]
- National Natural Science Foundation of China [81123001, 81570526]
- Innovation project [16CXZ027]
- Chinese State High-tech Program (863 Program) [2012AA020204, 2014AA020906]
- Program of International ST Cooperation [2014DFB30020, 2014DFB30010]
- Natural Science Foundation of Beijing [7152036]
- Open Project Program of the State Key Laboratory of Proteomics (Academy of Military Medical Sciences) [SKLP-O201509]
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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.
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