Developing a Bimolecular Affinity Purification Strategy to Isolate 26S Proteasome Holocomplexes for Complex-Centric Proteomic Analysis

The
26S proteasome is a mega-dalton protein complex responsible
for intracellular degradation in eukaryotes. It is composed of two
subcomplexes: the 20S core particle and the 19S regulatory particle,
which form compositionally and structurally heterogeneous proteasome
complexes in cells. To fully characterize the 26S proteasome, it is
necessary to understand its structural and functional diversities.
Multiple mass spectrometry (MS) methodologies have been developed
in recent years for the study of proteasome structural dynamics in
which biochemically isolated complexes are subjected to analysis.
Due to the inherent heterogeneity of proteasome complexes, single-bait
affinity purification typically results in a mixture of compositionally
heterogeneous complexes regardless of the baits, making accurate assessment
of complex-specific conformations and functions challenging. To facilitate
complex-centric analysis, we have adopted a bimolecular affinity purification
method utilizing a dual-bait cell line expressing tagged 19S and tagged
20S subunits to improve the homogeneity of the resulting 26S holocomplexes.
To establish the method, four types of purifications were performed
and the resulting samples were extensively examined by biochemical
analysis and two label-free quantitative MS methods. Our results have
demonstrated the effectiveness of this purification strategy in improving
the complex homogeneity for downstream biochemical and MS characterizations.
This strategy will be valuable for facilitating detailed quantitative
assessments of complex-specific molecular details under different
conditions and can be directly adopted for studying other complexes.