Dataset Information

Native mass spectrometry interrogation of complexes formed during targeted protein degradation.

ABSTRACT:

Rationale

Protein degraders are small molecules that promote cellular degradation of a target protein. Degraders simultaneously bind to their target and an E3 ligase, bringing them into close spatial proximity, but the formation of this ternary complex is difficult to measure with many biophysical techniques.

Methods

Native mass spectrometry (nMS) is an effective label-free technique to identify the complexes formed by proteolysis-targeting chimeras (PROTACs). It can monitor the formation of ternary E3-PROTAC-target complexes and detect intermediate binary species. Experiments are described using a Synapt G2Si (Waters) equipped with a nano-electrospray ionisation source.

Results

The protocol describes nMS experiments for measuring the complexes formed by PROTAC molecules. It also describes how to investigate differences in the affinity of PROTAC complexes, whether a PROTAC shows specificity for a given target and whether a PROTAC shows cooperative behaviour.

Conclusions

Here, we provide step-by-step instructions for the sample preparation of PROTAC complexes and their nMS interrogation to obtain optimal information on their binding modes.

SUBMITTER: Ahmed IMM

PROVIDER: S-EPMC10909470 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Publications

Native mass spectrometry interrogation of complexes formed during targeted protein degradation.

Ahmed Ikhlas M M IMM Beveridge Rebecca R

Rapid communications in mass spectrometry : RCM 20231101 22

<h4>Rationale</h4>Protein degraders are small molecules that promote cellular degradation of a target protein. Degraders simultaneously bind to their target and an E3 ligase, bringing them into close spatial proximity, but the formation of this ternary complex is difficult to measure with many biophysical techniques.<h4>Methods</h4>Native mass spectrometry (nMS) is an effective label-free technique to identify the complexes formed by proteolysis-targeting chimeras (PROTACs). It can monitor the f ...[more]

PMID: 37817340

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Project description:Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

Dataset Information

Native mass spectrometry interrogation of complexes formed during targeted protein degradation.

Rationale

Methods

Results

Conclusions

Publications

Native mass spectrometry interrogation of complexes formed during targeted protein degradation.

Similar Datasets

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