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Pull-down Assay on Streptavidin Beads and Surface Plasmon Resonance Chips for SWATH-MS-based Interactomics
Josef Maryáš1,2, Jakub Faktor1,2, Lenka Čápková1
1Masaryk University, Faculty of Science, Department of Biochemistry, Brno, Czech Republic.
Cancer Genomics & Proteomics
|September 9, 2018
Summary
Surface plasmon resonance (SPR) chips offer a reliable alternative to traditional pull-down assays for identifying protein interactions. This method, combined with mass spectrometry, enables reproducible characterization of cancer-associated interactomes.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Pull-down assays are widely used for identifying protein interactors in vitro.
- Characterizing protein-protein interactions is crucial for understanding cellular functions and disease mechanisms.
Purpose of the Study:
- To compare the efficacy of conventional pull-down assay variants with streptavidin-modified surface plasmon resonance (SPR) chips for detecting PDZ and LIM domain protein 2 (PDLIM2) interaction partners.
- To evaluate different methods for optimizing pull-down assays, including flow techniques and detergent concentrations.
Main Methods:
- Analysis of PDLIM2 protein-protein interactions using three pull-down assay variants on streptavidin beads.
- Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) in Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH) mode.
- Comparison of LC-SWATH-MS/MS data obtained from pull-down assays with data from SPR chips.
Main Results:
- SPR chips yielded data comparable to on-column streptavidin beads.
- Gravity flow and microflow methods in wash and elution steps outperformed centrifugation.
- Detergent type and concentration did not significantly impact the interactome data of PDLIM2.
Conclusions:
- SPR-based affinity purification coupled with SWATH mass spectrometry provides a reproducible and controlled method for characterizing interactomes.
- This approach is particularly valuable for studying cancer-associated proteins like PDLIM2.
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