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Integrated strategy for high-sensitive phosphoproteome analysis from low micrograms of protein samples


Phosphoproteomics has become popular proteomic technology for exploring cellular signaling networks. However, current approaches often require milligrams of protein samples which hamper their applications for translational purpose with limited starting materials. In this study, we aimed to challenge the lowest starting material limit for phosphoproteome profiling. By carefully optimizing the well-established high-pH reversed-phase (RP) fractionation plus Ti4+-IMAC enrichment strategy, we achieved the identification of 15260 and 8936 unique phosphopeptides from only 500 μg and 250 μg predigested peptides, respectively. To further improve the sensitivity of phosphoproteome analysis for low micrograms of protein samples, we developed an integrated strategy, termed Phospho-SISPROT. This technology integrates three tips in tandem for protein digestion by the simple and integrated spintip-based proteomics technology (SISPROT), phosphopeptide enrichment by Ti4+-IMAC tip, and desalting by StageTip, respectively, which could dramatically reduce the phosphoproteome analysis time from a couple of days to only 6 hours and improve the system sensitivity. The flow through of Phospho-SISPROT could be reused for the global protein identification, which is very helpful for accurate phosphoproteome analysis with limited starting materials. More than 5500 and 600 unique phosphopeptides were respectively identified from 20 μg and 1 μg pervanadate treated HEK 293T cell lysates processed by the Phospho-SISPROT. To the best of our knowledge, this performance is the highest record reported to date by using standard LC-MS/MS setup. We expect that the Phospho-SISPROT and the optimized high-pH RP fractionation plus Ti4+-IMAC enrichment strategy will be well suited for high-sensitive phosphoproteome analysis of rare biological samples.

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Publication details

The article was received on 28 Apr 2018, accepted on 09 Jun 2018 and first published on 11 Jun 2018

Article type: Paper
DOI: 10.1039/C8AN00792F
Citation: Analyst, 2018, Accepted Manuscript
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    Integrated strategy for high-sensitive phosphoproteome analysis from low micrograms of protein samples

    W. Chen, L. Chen and R. Tian, Analyst, 2018, Accepted Manuscript , DOI: 10.1039/C8AN00792F

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