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Updated: Jan 17, 2026
Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Published on: June 25, 2020
Fast and effective all-in-one single-tube phosphoproteomics workflow for high-throughput phosphoproteomics analysis
Dandan Zhou1, Cuntao Wei1, Yue Wang1
1Key Laboratory of Resource Biology and Modern Biotechnology in Western China, College of Life Science, Northwest University, Xi'an, Shaanxi Province, 710069, People's Republic of China.
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Phosphoproteomics is of great significance for deciphering the regulatory mechanisms of cell signaling. In order to overcome the limitations of current sample preparation methods, it is crucial to develop novel techniques that offer improved cost-effectiveness, ease of use, and reproducibility, while also reducing the reliance on hazardous reagents. We have innovatively developed a fast and effective all-in-one single-tube phosphoproteomics (FEAS-phospho) workflow, integrating protein extraction, digestion and phosphopeptide enrichment into a single-tube operation. TFA was used to efficiently extract proteins from cell and tissue samples within 3 min, without additional homogenization or sonication. By adjusting the enzyme-to-protein mass ratio, protein digestion time was reduced to from 16 h to 15 min. Meanwhile, cumbersome steps such as peptide desalting, lyophilization, and re-dissolution after digestion were omitted, allowing in-situ direct phosphopeptide enrichment from the protein digest, which provided an efficient and reliable solution for high-throughput phosphorylation analysis. This workflow greatly simplified the cumbersome operations in classic phosphoproteomics analysis, and the entire process from protein extraction to phosphopeptide enrichment could be completed within 2.5 h. With a micro-flow LC-MS/MS system, 5087 phosphorylation sites, 6247 phosphopeptides, corresponding to 2295 phosphoproteins were identified from 200 μg of MCF-7 protein digest under data-dependent acquisition (DDA) mode with a 15 min gradient. While the data-independent acquisition (DIA) mode identified more than 8400 phosphopeptides with the same quantity and gradient. The FEAS-phospho was further validated by phosphoproteome analysis of five different mouse tissue. The FEAS-phospho can be easily integrated into the automated phosphoproteomics workflow, and is expected to have broad applications in phosphoproteomics analysis.
