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A basic phosphoproteomic-DIA workflow integrating precise quantification of phosphosites in systems biology

Biophysics Reports 2023年第2期9卷 82-98页

作者： Yi Di Wenxue Li barbora salovska Qian Ba Zhenyi Hu Shisheng Wang Yansheng Liu Cancer Biology Institute Yale University School of Medicine Laboratory Center Shanghai Municipal Hospital of Traditional Chinese Medicine Shanghai University of Traditional Chinese Medicine 3. Department of Pulmonary and Critical Care Medicine and Proteomics-Metabolomics Analysis Platform West China Hospital Sichuan University Department of Pharmacology Yale University School of Medicine

Phosphorylation is one of the most important post-translational modifications(PTMs) of proteins, governing critical protein functions. Most human proteins have been shown to undergo phosphorylation,and phosphoproteomic studies have been widely applied due to recent advancements in highresolution mass spectrometry technology. Although the experimental workflow for phosphoproteomics has been well-established, it would be useful to optimize and summarize a detailed, feasible protocol that combines phosphoproteomics and data-independent acquisition(DIA), along with follow-up data analysis procedures due to the recent instrumental and bioinformatic advances in measuring and understanding tens of thousands of site-specific phosphorylation events in a single experiment. Here,we describe an optimized Phos-DIA protocol, from sample preparation to bioinformatic analysis, along with practical considerations and experimental configurations for each step. The protocol is designed to be robust and applicable for both small-scale phosphoproteomic analysis and large-scale quantification of hundreds of samples for studies in systems biology and systems medicine.

关键词： Bioinformatic analysis High-resolution mass spectrometry technology Phosphoproteomics Systems biology Systems medicine

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