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Biophysics Reports 2023, 9(2): 82-98 https://doi.org/10.52601/bpr.2023.230007
Protocol | Open Access | Issue | Published: 30 April 2023

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

Show Author's Information Yi Di1,,  Wenxue Li1,,  Barbora Salovska1,,  Qian Ba1,2,  Zhenyi Hu1,  Shisheng Wang3,  Yansheng Liu1,4( )
Cancer Biology Institute, Yale University School of Medicine, West Haven, CT 06516, USA
Current address: Laboratory Center, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China
Department of Pulmonary and Critical Care Medicine, and Proteomics-Metabolomics Analysis Platform, West China Hospital, Sichuan University, Chengdu 610041, China
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA

Yi Di, Wenxue Li and Barbora Salovska contributed equally to this work.

Keywords:
Systems biology, Bioinformatic analysis, Phosphoproteomics, High-resolution mass spectrometry technology, Systems medicine
Cite this article:
Di Y, Li W, Salovska B, et al. A basic phosphoproteomic-DIA workflow integrating precise quantification of phosphosites in systems biology. Biophysics Reports, 2023, 9(2): 82-98. https://doi.org/10.52601/bpr.2023.230007
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Abstract Full text About this article

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 high-resolution 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.


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About this article

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

Show Author's information Yi Di1,, Wenxue Li1,, Barbora Salovska1,, Qian Ba1,2, Zhenyi Hu1, Shisheng Wang3, Yansheng Liu1,4( )
Cancer Biology Institute, Yale University School of Medicine, West Haven, CT 06516, USA
Current address: Laboratory Center, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China
Department of Pulmonary and Critical Care Medicine, and Proteomics-Metabolomics Analysis Platform, West China Hospital, Sichuan University, Chengdu 610041, China
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA

Yi Di, Wenxue Li and Barbora Salovska contributed equally to this work.

Abstract

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 high-resolution 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.

Keywords: Systems biology, Bioinformatic analysis, Phosphoproteomics, High-resolution mass spectrometry technology, Systems medicine

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Received: 09 April 2023
Accepted: 28 April 2023
Published: 30 April 2023
Issue date: April 2023

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