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Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Published on: July 31, 2011
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Considerations for Identifying Endogenous Protein Complexes from Tissue via Immunoaffinity Purification and
Joel D Federspiel1, Ileana M Cristea2
1Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Methods in Molecular Biology (Clifton, N.J.)
|April 14, 2019
Summary
This study presents a protocol for identifying protein complexes using endogenous immunoaffinity purification and mass spectrometry. This method avoids artifacts from tagged proteins, enabling more accurate analysis of native protein interactions.
Area of Science:
- Molecular Biology
- Biochemistry
- Proteomics
Background:
- Protein complexes are crucial for biological processes, and identifying their composition provides insights into cellular functions.
- Affinity purification coupled with mass spectrometry (AP-MS) is widely used for protein-protein interaction studies, but often relies on tagged and overexpressed proteins, which can introduce artifacts.
- Existing AP-MS methods using tagged proteins can lead to inaccurate results due to the tag's presence and altered protein abundance.
Purpose of the Study:
- To present an optimized protocol for isolating and identifying endogenous protein complexes.
- To overcome challenges in endogenous protein isolation, such as antibody selection and experimental condition optimization.
- To enable the study of protein interactions in native cellular contexts without the artifacts associated with tagged proteins.
Main Methods:
- Optimization of lysis conditions and antibody evaluation for effective immunoaffinity purification.
- Application of quantitative mass spectrometry for identifying protein complexes from endogenous samples.
- Utilizing targeted mass spectrometry, such as parallel reaction monitoring (PRM), for orthogonal validation of identified interactions.
Main Results:
- A robust protocol for endogenous immunoaffinity purification of protein complexes.
- Successful identification of protein complex compositions using quantitative mass spectrometry.
- Demonstration of PRM as a valuable tool for validating protein interactions identified by data-dependent mass spectrometry.
Conclusions:
- Endogenous immunoaffinity purification coupled with mass spectrometry is a powerful approach for studying native protein complexes.
- This method provides a more accurate understanding of protein-protein interactions and cellular processes.
- The presented protocol facilitates the reliable identification and validation of protein complexes in various biological systems.
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