Native mass spectrometry interrogation of complexes formed during targeted protein degradation

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.


| INTRODUCTION
Proteolysis-targeting chimeras (PROTACs) are small-molecule degraders that eliminate target proteins by programming them for degradation by the cell. 1,2PROTACs are bifunctional molecules that consist of two ligands joined by a linker region. 3The ligands are specific for a target protein and an E3 ligase respectively. 4The PROTAC therefore brings the two proteins into close spatial proximity, causing the E3 ligase to catalyse ubiquitination of the target protein, thereby labelling it for proteasomal degradation by the cell. 5The term 'target protein', which is also referred to as the 'substrate' or 'protein of interest', will be used throughout the text.
A main challenge in studying PROTACs is the ternary binding system that involves the E3 ligase, the target protein and the PROTAC.Many biochemical methods have been developed to measure binary systems, often requiring adaptations and approximations to study ternary complexes.7][8] Native mass spectrometry (nMS) is a highly applicable method to study protein complexes formed by PROTACs as it reports on multiple binding stoichiometries present in dynamic protein mixtures, including species populated to a low extent.This sensitive, label-free method can be applied to proteins of varying mass and polydispersity, i.e. different shapes and sizes.
Protocols detailing nMS experiments are available that describe analysis of large protein complexes, 9 analysis of monoclonal antibodies 10 and direct characterisation of overproduced proteins, 11 among others.We have recently demonstrated that nMS can report on the formation of ternary complexes, determine the binding specificity of a PROTAC and compare complex formation with multiple target proteins in a single measurement. 12hilst this protocol was developed for PROTACs, it is suitable for the analysis of other protein degraders such as molecular glues 13,14 (as demonstrated by Bellamy-Carter et al 15 ), SNIPERS 16 and other bifunctional molecules. 17 Ammonium acetate is normally the solution of choice for nMS experiments, as it is highly volatile and hence evaporates from the proteins readily during desolvation.As the PROTAC will likely be dissolved in DMSO or another organic solvent, ensure that the proteins can be analysed in the presence of this and be aware of any alterations that it confers to the protein signal (i.e.charge reduction in the presence of DMSO 18,19 ).Moreover, keep the final DMSO/organic solvent concentration as low as possible; in this protocol the DMSO concentration is kept at 1% and it is not recommended to exceed 5%.• If MS signal is low for the proteins, the concentration of ammonium acetate could be altered to achieve optimal solution conditions.• Ammonium bicarbonate can also be used as an alternative solution.

Time Step Comments/tips
Micro bio spin chromatography columns 15-30 min • Invert the column sharply several times to resuspend the settled gel and remove any bubbles.Snap off the tip and place the column in a 2.0 mL microcentrifuge tube (included).Now remove the top cap.If the column does not begin to flow, push the cap back on the column and then remove it again to start the flow.Allow the excess packing buffer to drain by gravity to the top of the gel bed (about 2 min).Discard the drained buffer then place the column back into the 2.0 mL tube.
• These instructions are adapted from the instruction manual for micro Bio-Spin Chromatography columns, which can be found on bio-rad.com.

(Continues)
If there is a severe loss of protein concentration when using the spin columns, or if the protein target is intrinsically disordered and/or prone to aggregation, then microdialysis devices can be used as an alternative.Molecules smaller than the device's exclusion limit will be in the dialysis buffer (see specifications).
• These instructions are adapted from the instruction manual for Pierce 96-well microdialysis plate, which can be found on thermofisher.com.• Ensure the samples are measured quickly (within 30 min of the addition of the reagent) as the method is time-sensitive.• The absorbance readout for the samples should be within the calibration range (i.e.1-10 μg).If not, the concentration assay will need to be repeated with diluted samples.

Time
Step

| Protein quality control and determination of optimum protein concentrations
This step is to measure the mass of the individual proteins and assess their purity.
2.2.1 | Analyse the proteins individually for quality control, to optimise concentrations and to measure their mass

| Test for ternary and binary complex formation
This step is to identify whether ternary complexes are formed with a given E3-PROTAC-target combination.Identification of which charge states the complex is present in (see Data S1) will also help with data analysis at further points in the protocol (e.g.Section 2.6).In these spectra, the presence of binary interactions (PROTAC-E3 and PROTAC-target) in the reaction mixture can also be identified.which requires low sample volumes, low concentrations and is a soft ionisation method.We recommend using metalcoated borosilicate capillaries (Thermo Scientific).Example settings for the mass spectrometer are provided in Section 2.7.• Determine the isotope-averaged molecular mass of the proteins, using a deconvolution software such as Origami 20 or Unidec. 21For nESI-MS, 10 μL per sample is required.
• If signal intensity is low, try increasing or decreasing the protein concentration.Decreasing the protein concentration can have a positive effect on the signal intensity, as competitive ionisation can occur at higher concentrations. 22I G U R E 1 Characterising the binding equilibrium between the E3 ligase (5 μM), the proteolysis-targeting chimera (PROTAC) and the target (5 μM) by nMS in the absence of PROTAC (A) and in the presence of PROTAC (10 μM) (B). Figure 1B shows a spectrum of the same target and E3 as in Figure 1A, with the addition of 10 μM PROTAC.In this case, the ternary complex (highlighted in pink) and target-PROTAC and E3-PROTAC binary complexes are observed.

| Test for specificity
PROTACs can recruit closely related target proteins to different extents, 7 and the specificity of a PROTAC can be estimated by nMS by comparing the relative intensity of the ternary complex to that of the unbound E3 ligase (Figure 2A).

| Test cooperativity
In a cooperative system, the ternary complex will form more readily than either of the binary complexes, which is thought to contribute to the specificity of the PROTAC.Cooperativity can be measured by comparing binary complex formation to ternary complex formation.In a cooperative system, binary complexes between the PROTAC and either protein will be low (Figure 2B).

Time
Step Comments/tips

15-30 min
• Analyse target protein-PROTAC and E3-PROTAC separately to measure binary complex formation.• Compare ternary complex formation to binary complex.If the signal intensity for ternary complexes is higher than for binary complexes, the ternary complex formation is likely cooperative.
• For in-depth study, analyse binary complex (PROTAC-target or PROTAC-E3) in the presence of increasing concentrations of the third component and monitor the ternary complex formation.

| Develop competition assays
To take full advantage of the ability of nMS to measure complex reaction mixtures, assays can be developed in which the specificity for multiple target proteins can be measured in a single experiment.
Measuring the target proteins in mixtures is more time-effective than separate measurements and has the added advantage of providing information on competition between targets forming the ternary complexes (Figure 2C).

• 5 •
Dissolve 0.5 mg of BSA in 0.5 mL of water to prepare 1 μg/μL BSA.•Prepare standards in microcentrifuge tube (1.5 mL) as follows: Estimate sample protein volume for 1, 5 and 10 μg.Protein dilution might be required.• Add the appropriate volumes of sample protein to 0.5 mL water/0.5 mL Bradford reagent.• Mix by inverting the microcentrifuge tube, and transfer samples to disposable UV-visible cuvettes.•Measure absorbance at 595 nm using blank for zeroing the reading.•Prepare a scatter graph for BSA standards: A 595nm (Y-axis) versus BSA mass/μg (X-axis).The R 2 value should be above 0.9; record the m and c values from the graph (y = mx + c).• Concentration of sample (mg/mL) = [(sample absorbance + (Àc)/m)/(μL of added protein sample)].• Take average of all three readings for final concentrations.

Figure•
Figure 1A shows an example mass spectrum of a target and E3 in the Figure adapted from Beveridge et al. 12 [Color figure can be viewed at wileyonlinelibrary.com] protein, highlighted in blue, is present in charge states 6+ to 19+, with most of the intensity in the 6+ and 7+ charge states.The E3 ligase, highlighted in green, is present in charge states 9+ to 12+.

F•
I G U R E 2 nMS for specificity (A), cooperativity (B) and competition assay studies (C).[Color figure can be viewed at wileyonlinelibrary.com]Analyse a mixture of potential targets at equal concentration to the E3 ligase, with the optimal amount of PROTAC identified in Section 2.3, to identify which targets are recruited when there is no competition for binding.• To measure which targets win competition for binding, increase the concentration of the targets relative to the E3 ligase.Higher relative signal intensity indicates the preferred target.