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Identification of protein complexes with quantitative proteomics in S. cerevisiae
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Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.

Mi Ke, Jie Liu, Wendong Chen

  • 1Shenzhen People's Hospital , The Second Clinical Medical College of Jinan University , Shenzhen 518020 , China.

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|October 4, 2018
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We developed an integrated Affinity Purification-Simple and Integrated Spintip-based Proteomics (AP-SISPROT) workflow for rapid and efficient protein complex analysis. This method enhances digestion efficiency and enables precise profiling of endogenous protein interactions.

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Area of Science:

  • Proteomics
  • Molecular Biology
  • Biochemistry

Background:

  • Proteins function in complexes, and Affinity Purification-Mass Spectrometry (AP-MS) is key for their study.
  • Traditional AP-MS workflows are time-consuming and can lead to incomplete protein digestion.

Purpose of the Study:

  • To develop a rapid, integrated AP-MS workflow for efficient protein complex analysis.
  • To improve digestion efficiency and enable precise quantification of protein interactions.
  • To facilitate the study of endogenous protein complexes using CRISPR Cas9 technology.

Main Methods:

  • Integration of Affinity Purification (AP) with Simple and Integrated Spintip-based Proteomics (SISPROT) technology.
  • Development of a quantitative data analysis workflow for distinguishing true interactors from non-specific binding.
  • Application of CRISPR Cas9 for generating endogenously expressed, tagged bait proteins.

Main Results:

  • The AP-SISPROT workflow processes over 10 samples in 2 hours with high digestion efficiency, particularly for large transmembrane proteins like EGFR.
  • Accurate temporal profiling of the GRB2 protein interaction network was achieved.
  • Endogenous protein complexes, such as those involving Integrin-linked Kinase (ILK), were identified with higher selectivity compared to overexpressed proteins.

Conclusions:

  • The AP-SISPROT workflow offers a significantly faster and more efficient method for protein complex analysis.
  • Combining AP-SISPROT with CRISPR Cas9 enables the study of endogenous protein complexes with high physiological relevance and selectivity.
  • This integrated approach is broadly applicable for advancing the understanding of protein complex dynamics.