Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes

Area of Science:

• Molecular Biology
• Biochemistry
• Cancer Research

Background:

• The Epidermal Growth Factor Receptor (EGFR) plays a critical role in various cancers, particularly lung cancer.
• Understanding EGFR signaling pathways involves identifying its interacting protein partners.
• Tandem Affinity Purification (TAP) is a valuable technique for protein complex identification.

Purpose of the Study:

• To downscale the SH-TAP method for efficient protein complex identification using low input material.
• To identify the major protein constituents of EGFR deletion mutant and Grb2 complexes.
• To provide guidelines for optimizing TAP experiments with limited protein quantities.

Main Methods:

• Stable retroviral expression of tagged EGFR deletion mutant (L747-S752) in PC9 lung cancer cells.
• Stable doxycycline-inducible expression of tagged Grb2 in HEK293 cells using Flp-mediated recombination.
• Downscaling SH-TAP to 5-12.5 mg total protein input and subsequent mass spectrometry analysis.

Main Results:

• Successfully identified major constituents of the EGFR del complex (e.g., GRB2, ERRFI) and Grb2 complex (e.g., SOS1, CBL).
• Demonstrated successful protein complex identification at significantly reduced input levels (0.5-1 × 15 cm culture plate).
• Determined that optimizing protein quantity injected into mass spectrometry is crucial for sequence coverage and identifying interacting proteins.

Conclusions:

• The downscaled SH-TAP method is effective for identifying protein complexes from low input material.
• This optimized technique facilitates the study of protein interactions in systems with limited cellular material.
• The findings provide practical guidance for researchers performing TAP, especially with scarce samples.