Identification of Ligands for Ion Channels: TRPM2

Transient receptor potential melastatin 2 (TRPM2) is a calcium‐permeable, nonselective cation channel with a widespread distribution throughout the body. It is involved in many pathological and physiological processes, making it a potential therapeutic target for various diseases, including Alzheimer's disease, Parkinson's disease, and cancers. New analytical techniques are beneficial for gaining a deeper understanding of its involvement in disease pathogenesis and for advancing the drug discovery for TRPM2‐related diseases. In this work, we present the application of collision‐induced affinity selection mass spectrometry (CIAS‐MS) for the direct identification of ligands binding to TRPM2. CIAS‐MS circumvents the need for high mass detection typically associated with mass spectrometry of large membrane proteins. Instead, it focuses on the detection of small molecules dissociated from the ligand‐protein‐detergent complexes. This affinity selection approach consolidates all affinity selection steps within the mass spectrometer, resulting in a streamlined process. We showed the direct identification of a known TRPM2 ligand dissociated from the protein‐ligand complex. We demonstrated that CIAS‐MS can identify binding ligands from complex mixtures of compounds and screened a compound library against TRPM2. We investigated the impact of voltage increments and ligand concentrations on the dissociation behavior of the binding ligand, revealing a dose‐dependent relationship.


Introduction
The transient receptor potential melastatin 2 (TRPM2) is a Ca 2 + permeable non-selective cation channel, within the transient receptor potential (TRP) ion channel superfamily. [1]The TRPM2 channel protein is characterized by six transmembrane domains (S1-S6) with a pore-forming loop located between S5 and S6 and multiple functional domains in the cytoplasmic tails (Figure 1). [2]TRPM2 exhibits a widespread distribution throughout the human body and participates in many physiological processes, including apoptosis, heat sensing, and immune response. [3]The channel is considered a potential therapeutic target for neurological diseases such as cerebral ischemia, Alzheimer's disease, and Parkinson's disease. [2,4]Additionally, TRPM2 is also implicated in other diseases, including diabetes, acute kidney injury, acetaminophen-induced liver damage, and various cancers. [5]The activation of TRPM2 is closely associated with oxidative stress and can be triggered by stimuli such as adenosine diphosphate ribose (ADPR), intracellular Ca 2 + , and hydrogen peroxide (H 2 O 2 ). [1,6]mbrane proteins comprise more than 60 % of the current drug targets, with ion channels like TRPM2 accounting for approximately 19 % of these. [7]However, investigation into membrane proteins is fraught with challenges due to their amphipathic nature, the complexities associated with their expression and purification, and their limited natural abundance. [8]The development and application of novel analytical techniques can be beneficial for better comprehending the involvement of these significant drug targets in diseases, and expediting the discovery of potential therapeutics.One such method that can be applied is native mass spectrometry (MS), with studies demonstrating its ability to probe membrane protein-lipid interactions. [9]Native MS has been proven capable of revealing thermodynamics and allostery of individual lipid binding events. [10]ative-omics is an approach integrating native MS with small molecule fragmentation, to identify bound ligands released after native MS. [11]Using a modified tribrid Orbitrap MS instrument, multiple stages of MS are conducted.After the ionization of detergent-encapsulated membrane proteins, an activation step is performed to liberate the protein-ligand complex from the detergent micelle encapsulating it.The complex is isolated and dissociated into proteoforms and ligands.The ligand is then fragmented for identification through database searching. Detecting a small molecule binding to the large ion channels, such as the TRPM2 ion channel, requires mass spectrometers with a high range transfer and detection.An empirical model of protein mass vs m/z value has been published with a typical expected m/z of around 4,000-12,000. [11]Even a monomeric TRPM2 with an estimated mass of ~170 KDa, will fall into the m/z range of > ~9,000. [13]In this case, a small molecule with a MW of 300 Da binding to the protein would appear as a m/z shift of ~16.Detection of small molecules with such small mass shift could be difficult without the appropriate instrument resolution and modification.
We recently introduced Collision-Induced Affinity Selection MS (CIAS-MS), a technique that relies on the detection of small molecules, significantly reducing the mass detection requirement (Figure 2A). [14]General AS-MS techniques, such as size exclusion AS-MS and pulsed ultrafiltration AS-MS, offer the advantage of not requiring the protein to be visible, but they involve multiple preparation steps prior to the MS analysis to identify a bound ligand (Figure 2B). [15]In contrast, CIAS-MS eliminates the need for pre-screening external removal of unbound compounds by integrating all processes into a single MS experiment.CIAS-MS employs the same electrospray injection for ionizing the sample as in native MS.The ionized mixture is transferred into the quadrupole for mass selection, allowing for the capture of the protein-ligand complexes while eliminating small unbound molecules.Subsequently, collisionalinduced dissociation (CID) is used to dissociate the proteinligand complex.The dissociated protein and ligand are then transferred to the ion guide, featuring a low time-of-flight (TOF), which permits only the small ligand to enter the mass analyzer for detection (Figure 2A). [14]CIAS-MS has previously been applied to screen a mixture of compounds against a soluble protein, SARS-CoV-2 non-structural protein 9 (nsp9). [14]o better address the challenge in ion channel ligand identification, we applied CIAS-MS for screening against TRPM2.We first utilized TRPM2 and its known inhibitor clotrimazole in the proof-of-concept experiment.Using CIAS-MS, we detected clotrimazole dissociated from TRPM2-clotrimazole complex.We demonstrated that CIAS-MS methods could identify clotrimazole bound to TRPM2 in a mixture of 100 compounds.TRPM2 was also screened against a natural product library containing 2000 compounds, with a binder identified.The impact of various collisional voltages and ligand concentrations on the dissociation behavior of the ligand was also explored.

Expression and Purification of TRPM2
Following the transient transfection of TRPM2-expressing construct (pEF6/V5-His_hTRPM2) into Expi293F cells, the His-tagged TRPM2 proteins were solubilized with dodecylmaltoside (DDM) and purified using immobilized metal affinity chromatography (IMAC) technique.The solubilization and purification of TRPM2 were monitored through Western blot analysis.As shown in Figure 3, TRPM2 proteins can be visualized as a prominent band at appoximately 170 kDa.The TRPM2 band observed in the solubilized TRPM2 sample prior to IMAC (Sol) confirmed the successful extraction and solubilization of the channel protein, as any insoluble material was removed during the purification procedure.Western blot analysis also confirmed the successful IMAC elution of TRPM2 (elution 1-4) without passing through in the wash steps (wash 1-3).

CIAS-MS of TRPM2 Binding to Clotrimazole
Detergent micelles are the most frequently used membrane mimetics for mass spectrometry of membrane proteins, with prior studies demonstrating the transfer of protein-DDM micelle complex into the gas phase while preserving ligand binding. [16]fter transferring the membrane proteins encapsulated within the micelles into the mass spectrometer, the detergent can be removed by applying activation energy. [17]CID of the collisional cell and skimmer 1 (situated at the end of funnel 2), are the regions where activation energy can be manipulated in a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer. [18]Lippens and coworkers have previously investigated the impact of these FT-ICR MS parameters on the analysis of membrane proteins, showing that the high resolution of FT-ICR platform allows the observation of low molecular weight modification on membrane proteins. [18]n the present study, CIAS-MS was applied to observe dissociated clotrimazole from TRPM2-clotrimazole complexes on a FT-ICR platform.Clotrimazole, an imidazole antifungal agent with applications in treating fungal infections and explored as a potential therapeutic option for sickle cell disease, has been identified as a TRPM2 ligand inhibiting ADPR-induced TRPM2 activity with an IC 50 of 3-30 μM in electrophysiology studies. [19]It was also reported that clotrimazole inhibits the H 2 O 2 -induced TRPM2 current in CA1 rat hippocampal neurons. [20]Additionally, pharmacological inhibition of TRPM2 by clotrimazole revealed a significant reduction in oxygenglucose deprivation-induced cell death in neurons from male mice. [21]imilar to native MS of membrane proteins, the CIAS-MS process commenced by ionizing the protein-detergent complex and the transmission into the MS.Skimmer 1 was used to aid ion transmission and in-source dissociation.Following which, the quadrupole was employed for mass selection, trapping the larger protein-ligand complexes while removing smaller unbound molecules.The captured complexes were transferred to the collision cell for collisional-induced dissociation, which facilitates the liberation of the complexes from the micelle and initiates dissociation of the bound ligand.Control experiments were completed to verify the specificity of the CIAS-MS method by injecting 10 μM TRPM2 protein or 10 μM clotrimazole only, both with and without CID enabled were performed (Figure 4A-D).No ions were detected within the mass range of m/z 200-2000 in the spectra of control experiments.Fragmented DDM molecules with a potassium adduct were observed in the spectrum of TRPM2 protein after the activation of CID (Figure 4B).The results from the control experiments demonstrates that the current mass selection settings excluded any unbound small molecules.
10 μM TRPM2 incubated with 10 μM clotrimazole was transferred into the MS.In the absence of CID, no ions were observed within the selected mass range (Figure 4E).With CID enabled, an ion signal was detected at m/z 277.08, corresponding to a fragment ion ([M-C 3 H 4 N 2 + H] + ) of clotrimazole resulting from the loss of the imidazole moiety, aligning with previously reported spectra data (Figure 4F). [22]The MS spectrum of clotrimazole after positive electrospray ionization resulted in ion signals for a hydrogen adduct at m/z 345.11, accompanied by a 37 Cl isotope peak at m/z 349.11, as well as a 61 % [M-C 3 H 4 N 2 + H] + ions at m/z 277.08, accompanied by a 37 Cl isotope peak at m/z 279.07, which confirmed this fragmentation (Figure S1).The results indicate that CIAS-MS can transfer the TRPM2-clotrimazole-detergent complex into the gas phase, excluding unbound molecules using the quadrupole analyzer, and enabling the dissociation and capture of the bound ligand from the ligand-protein complex for detection.

CIAS-MS for Spiked Clotrimazole against TRPM2
The capability of CIAS-MS to detect bound clotrimazole within a pooled compound mixture was assessed.10 μM TRPM2 was incubated with a natural product library pool containing 100 compounds, including 10 μM clotrimazole.Control experiments were conducted by injecting the pooled compound mixture alone, both with and without CID enabled, yielding no ion signals within the mass range (Figure 5A-B).When 10 μM TRPM2 incubated with the compound pool was infused into the mass spectrometer, no ions were detected without activating CID, suggesting unbound small molecules were exhausted to the vacuum (Figure 5C).When CID was activated, a fragment ion signal for clotrimazole at m/z 277.08 was observed.The [M + H] + ion was not observed, possibly due to the addition of collision energy (Figure 5D).The application of CIAS-MS to identify bound ligand in a compound mixture of nine compounds incubated with a soluble protein, non-structural protein 9 (nsp9) was previously shown. [14]In the current study, we extended the application of CIAS-MS to demonstrate that it can be applied to detect a bound TRPM2 ligand, clotrimazole, within an incubation mixture containing 100 compounds.

Natural Product Library Screening using CIAS-MS
A natural product library screening against TRPM2 was conducted using CIAS-MS.The library comprised 2000 compounds with 100 compounds in each pool (pool A-V), and each compound was present at a concentration of 10 μM.Control experiments were performed for each library pool alone, both with and without the activation of CID, ensuring that the current mass selection range efficiently removed any unbound molecules (the control experiment for the pool with the identified hit can be found in Figure S2).When CID was enabled, ion signals were detected in pool D, with the most pronounced signal at m/z 809.44 (Figure 6A).The hit was identified as ginsenoside F3, which appeared as the base peak of a potassium adduct at m/z 809.44, along with m/z 771.49 and 793.47, corresponding to ginsenoside F3 with a 18 % hydrogen adduct and a 12 % sodium adduct, respectively.Ginsenoside F3 is a saponin found in Panax ginseng and is known for its potent protective effect against ROS-induced oxidative stress (Figure 6B). [23]Intriguingly, numerous studies have consistently implicated the involvement of TRPM2 in the response to oxidative stress. [24]To further validate the accuracy of the current approach, 10 μM nsp9, the protein previously utilized in the CIAS-MS proof-of-concept study, was incubated with pool D at the same concentration, and subsequently subjected to CIAS-MS.No ion signals were detected, indicating the absence of ligand binding to nsp9 (Figure 6C and D).This was further confirmed by native MS of nsp9 incubated with pool D, where no sufficient protein-ligand complex was observed, particularly in relation to ginsenoside F3 (Figure S3).CIAS-MS of 10 μM TRPM2 incubated with 10 μM ginsenoside F3 was also performed to confirm the binding.Similar to the observation in TRPM2 incubated with pool D, ion signals were observed for the potassium adduct at m/z 809.45, for the hydrogen adduct at m/z 771.49 and the sodium adduct at m/z 793.47.In positive ionization mode, ginsenoside F3 appeared as the base peak with a m/z of 809.44, corresponding to the potassium adduct, and a hydrogen adduct at m/z 771.49, which accounted for 47 % of the ion species (Figure S1B).Following the activation of CID, the abundance of the hydrogen adduct species decreased, while sodium adduct species became apparent, suggesting that adduct ions were formed in the collision cell (Figure 6E).

Comparison of Ligand Dissociation Behaviors at Different Concentrations
Evaluation of the interactions between binding partners is crucial for gaining insights into biochemical mechanisms and the effectiveness of bioactive molecules.Mass spectrometrybased approaches for the assessment of binding affinities are usually conducted through titration, competition or melting curve experiments. [25]Titration experiments determine the  equilibrium binding constants through incubation of the two binding partners, where the concentration of one binding partner, often the protein, is held constant, while the concentration of the other is varied. [26]Competition measurements rely on the competition between a binding ligand and a reference ligand, both of which interact with the same binding site.These experiments then monitor changes in equilibrium between the reference ligand and the protein target. [27]25d] In addition to these conventional methods, laser-induced liquid bead ion desorption (LILBID) mass spectrometry offers an alternative approach for the quantitative determination of binding affinities. [28]28b] The main difference between CIAS-MS and LILBID-MS lies in the source of the energy input.LILBID-MS uses an infrared laser pulse, while CIAS-MS relies on the CID acceleration voltage to induce collision with the collision gas.The energy directed to induce the dissociation of the binding ligands in CIAS-MS can be altered in a manner similar to that in LILBID-MS.This allows for control over the degree of the dissociation by regulating the amount of energy directed to the sample.Weakly bound complexes with poor stability are more susceptible to ligand dissociation than strongly bound complexes after transferring into MS. [29]Under CIAS-MS conditions, complexes stabilized by higher-affinity ligands can withstand higher CID voltage.Additionally, when a greater quantity of protein-ligand complexes is formed due to an increase in ligand concentration, there will be more binding ligand available for dissociation.This higher ligand concentration can subsequently lead to a higher ion signal intensity when compared to a lower concentration.
In CIAS-MS, the ability to control the CID energy applied to the system, coupled with the observation that higher ligand concentrations can lead to increased dissociation, offers a valuable means to investigate the dissociation behavior of the clotrimazole-TRPM2 complex at different ligand concentrations.All MS parameters were kept constant, except for the applied CID voltage.The samples containing TRPM2 incubated with 10 μM or 100 μM clotrimazole were subjected to a voltage ramping of CID voltage, spanning 21 voltages from 0 V to 100 V, at 5 V intervals (Figure 7A).The resulting ligand dissociation is plotted in a voltage ramping dissociation curve, with signal-to-noise (S/N) ratio of the dissociated ligand plotted against the CID voltage.The voltage ramping curve included data prior to reaching a plateau.
In both samples, clotrimazole commenced dissociating at CID voltages above 15 V.This observation suggests that, under the current parameter settings, the minimum CID activation energy required to eject the complex from the detergent micelle and initiate the dissociation of the bound ligand was 15 V. TRPM2 incubated with 10 μM or 100 μM clotrimazole both maintained the packets of ions up to a CID voltage of 50 V.However, the increase in S/N ratio decelerated at a lower voltage increment of 40 V for 10 μM clotrimazole, compared to the continuous increase observed until the plateau voltage for 100 μM clotrimazole.The 100 μM clotrimazole sample consistently achieved a higher S/N ratio than the 10 μM clotrimazole sample throughout the elevated voltage increments.Particularly, the maximum S/N ratio for ligand dissociation observed at 50 V for the 100 μM clotrimazole sample was approximately double that of the 10 μM clotrimazole sample, indicating a dose-dependent relationship (Figure 7B and C).
19c] The correlation between voltage increments and ligand dissociation behavior, indicates that increased CID energy input to the system leads to more intense ligand dissociation from the complex.Higher CID energy is required for dissociating a larger amount of proteinligand complex.

Conclusions
Conventional AS-MS requires the external separation of the large protein-ligand complexes from the unbound molecules, the subsequent dissociation of the low molecular weight ligand from the complexes, and the final capturing and visualization of the dissociated ligand in MS.In contrast, CIAS-MS represents an affinity selection-based approach that streamlines all these steps within a single MS analysis, yielding time-saving benefits.
Here we demonstrated the visualization of dissociated clotrimazole from the clotrimazole-TRPM2 complex with the application of CIAS-MS.This observation of bound clotrimazole dissociated from the complex in MS-based studies has not been previously reported.19c] The higher S/N ratio observed for TRPM2 incubated with the higher concentration clotrimazole, compared to incubation with the lower concentration in the voltage ramping experiment confirmed this concentration-dependent effect.Additionally, a library screening on TRPM2 was conducted, which led to the identification of a potential binder ginsenoside F3, a saponin found in Panax ginseng known for its anti-oxidative activity. [23]perimental Section

Molecular cloning
The TRPM2 gene amplification was carried out using polymerase chain reaction (PCR) with forward primer KpnI (5'-TTAATTGG-TACCGCCACCATGGAGCCCTCAGCCCTG-3') and reverse primer XbaI (5'-GCGCCGTCTAGAGCGTAGTGAGCCCC GAACTCAG-3') (New England Biolabs).The PCR product was subjected to double digestion with KpnI/XbaI restriction enzymes.The double digested sample was then gel-purified with GenElute Gel Extraction Kit (Sigma-Aldrich, Australia) and ligated into the pEF6/V5-His vector with a molar ratio of 1 : 5 for vector to insert.The vector contains a Cterminal V5 epitope for Western blot detection using anti-V5 antibody, and a C-terminal polyhistidine (6×His) tag for purification with a nickel column.The TRPM2 expression plasmid was transformed into Competent DH5α cells (Thermo Fisher Scientific, USA).Isolation of plasmid DNA was performed using PureLink HiPure Plasmid Midiprep Kit (Invitrogen, USA).The resulting plasmid was validated by DNA sequencing.

Cell culture and transfection
Expi293F cells (Gibco, USA) were maintained in suspension culture in 125 mL Erlenmeyer flasks (Thermo Fisher Scientific, USA) using serum-free suspension Expi293F Expression Medium (Gibco, USA).The flasks were placed on an orbital shaker operating at a speed of 120 � 5 rpm, at 37 °C with 8 % CO 2 .Routine subculturing was carried out when the cell density reached 3-5×10 6 viable cells/mL.Transfections were exclusively conducted with cells exhibiting viability exceeding 90 %, and cells beyond passage number 20 were excluded.On the day prior to transfection, the cells were split to a density of 2.5-3×10 6 cells/mL.On the day of transfection, the cells were diluted to a final density of 3×10 6 cells/mL.Transient transfection was conducted using the ExpiFectamine 293 Transfection Kit (Gibco, USA).ExpiFectamine293 diluted in Opti-MEM (Gibco, USA) was added to plasmid DNA at a concentration of 1.0 μg/mL of culture volume.The mixture was transferred into each flask after 5 min incubation at room temperature.At 20 hours post-transfection, enhancers 1 and 2 were supplemented into the medium.The cells were harvested 4 days post-transfection by centrifugation at 500×g for 10 minutes and washed with buffer containing 50 mM Tris base (pH 8), 150 mM NaCl, 1 mM PMSF, and 1× complete protease inhibitor cocktail (Roche, USA).

Cell lysis and solubilization of TRPM2
The cell pellets were subjected to sonication using a Branson 450 Sonifier (Branson, USA), with three 10 second cycles.The lysed cells were centrifuged at 15,000×g at 4 °C for 30 minutes, and the supernatant free of cellular debris and unlysed cells was collected.The sample was then subjected to ultra-centrifugation with a Sorvall WX Ultra Series ultra-centrifuge (Thermo Fisher Scientific, Australia) at 200,000×g at 4 °C for 2 hours.The supernatant containing soluble protein fractions were removed, while the membrane pellet was resuspended in a solubilization buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 10 % glycerol, 1 mM PMSF, 1×complete protease inhibitor cocktail, and 1 % DDM (Avanti, USA).The solubilization was performed at 4 °C overnight by agitation with a magnetic stir bar.A second round of ultracentrifugation was conducted at 200,000×g at 4 °C for 1 hour to remove any remaining insoluble material.The supernatant, containing the solubilized membrane proteins, was collected for purification.

Immobilized metal affinity chromatography purification
10 mM imidazole was added to the solubilized membranes prior to column binding.Immobilized metal affinity chromatography (IMAC) purification was performed using a spin column loaded with HisPur Ni-NTA resin (Thermo Fisher Scientific, Australia), which was equilibrated with two resin bed volumes of an equilibration buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 10 mM imidazole, and 0.05 % DDM.The solubilized membrane proteins with 10 mM imidazole added were allowed to bind to the column by incubating it on a rotatory platform at 4 °C overnight.Following column binding, the unbound material was removed by centrifugation at 700×g for 2 minutes.The resin was subjected to three wash steps (Wash 1-3) with two resin bed volumes of a wash buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 25 mM imidazole, and 0.2 % DDM.The TRPM2 protein was then eluted four times (Elution 1-4), each using one resin bed volume of the elution buffer comprising 50 mM Tris, 150 mM NaCl, 400 mM imidazole, and 0.05 % DDM.The concentration of purified TRPM2 was determined by measuring the A280 molar absorbance using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Australia).

Western blotting
Samples obtained from solubilization and purification steps were mixed with 1× NuPAGE LDS sample buffer and 1× NuPAGE sample reducing agent (Invitrogen, USA).The mixture was heated for 10 minutes at 50 °C.SDS-polyacrylamide gel electrophoresis was carried out using a NuPAGE 4 to 12 %, Bis-Tris gel.The gel was then transferred onto a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (Bio-Rad, Australia).Blocking of the membrane was conducted with 5 % BSA in 1× TBS-T (TBS + 0.1 % Tween-20) for 1 hour at room temperature.The membrane was incubated with rabbit anti-V5 antibody (1 : 1000, Cell Signaling Technologies, D3H8Q) for 2 hours at room temperature, and washed with 1× TBS-T for 3×5 min with shaking.Subsequently, the membrane was incubated with IRDye 680RD-conjugated Goat Anti-Rabbit IgG H&L preabsorbed secondary antibody (1 : 10000, Abcam, ab216777) for 1 hour at room temperature.The blot was visualized with the Odyssey CLx Imaging System and quantified using ImageStudio software (Li-Cor).

Sample preparation
IMAC purified TRPM2 was processed through an Amicon Ultra-0.5 100 kDa molecular-weight cut-off (MWCO) concentrator (Millipore) to eliminate excess DDM micelle.The sample was then subjected to buffer exchange into MS buffer comprising 200 mM ammonium acetate (pH 8) with 2× CMC DDM using a NAP-5 column (Cytiva, USA).For the CIAS-MS of TRPM2 against compounds or library pool, 90 μL of the protein working solution at 10 μM was added to each compound or a library pool dissolved in 10 μL of methanol and incubated at room temperature for 30 minutes.Nsp9 protein was produced as previously described and buffer-exchanged with 200 mM ammonium acetate (pH 7) using a NAP-5 column (Cytiva, USA). [30]

Compounds
Clotrimazole was purchased from Sigma-Aldrich while ginsenoside F3 was sourced from Compounds Australia.The natural product library was obtained from Compounds Australia.The library contained 20 pools prepared in DMSO with 100 compounds in each pool.Each pool was freeze-dried to remove the DMSO, and resuspended in 10 μL methanol, prior to the incubation with 90 μL of protein.The final screening concentration for each compound in the pools was 10 μM.

Instrument settings for CIAS-MS and native MS
The MS experiments were carried out using a Bruker SolariX XR 12 T Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics Inc., Billerica, MA).Data acquisition was conducted in a Windows operating system using SolariX control software for Bruker SolariX XR 12T.
The ESI source was operated in direct injection configuration at a flow rate of 220 μL/h with a 500 μL Hamilton syringe.The nebulizing gas used was nitrogen gas with a pressure of 2 bar.The capillary voltage, end-plate offset, dry gas flow rate and the temperature were set to 4000 V, À 500 V, 4 L/minute, and 200 °C, respectively.The source optics capillary exit was at 200 V and deflector plate was at 220 V.The voltage of funnel 1 and funnel 2 were kept at 150 V and 6 V, respectively.The applied voltages of skimmer 1 was 75 V, while skimmer 2 was 5 V.The quadrupole was set to capture ions with m/z above 2500.The collision gas flow, collisional RF amplitude, RF frequency were configured to 80 %, 1600 V pp and 1 MHz, respectively.Collisional induced dissociation (CID) was conducted by colliding ions with argon in the collision cell with collision voltages ranging from 0 V to 100 V as indicated.The time-of-flight (TOF) was set to 0.65 ms.
Native MS analysis of nsp9 was performed using ESI source configured for direct injection at a flow rate of 120 μL/h.The nebulizing gas nitrogen was kept at a pressure of 2 bar.The capillary voltage, end-plate voltage, dry gas flow rate and the temperature were set to 4000 V, À 500 V, 4 L/minutes and 200 °C, respectively.The source optics capillary exit was kept at 200 V, while deflector plate was kept at 220 V. Funnel 1 and skimmer 1 operated with voltages of 150 V and 30 V, respectively.Optical transfer frequency and TOF were at 2 MHz and 1.5 ms, respectively.

Figure 2 .
Figure 2. CIAS-MS strategy vs. AS-MS strategy.(A) In CIAS-MS, the sample mixture is directly injected into the MS following incubation.The direct observation of the ligand mass spectrum is achieved with all steps performed internally.(B) In AS-MS, the protein-ligand complexes formed following incubation is first separated from the unbound compounds.The bound ligand is dissociated, and another separation step is then conducted to capture the low molecular weight (MW) ligand.Lastly, the ligand is injected into the mass spectrometer for analysis.

Figure 3 .
Figure3.Western blot analysis confirming the successful expression, extraction, solubilization and purification of TRPM2 proteins.Ladder: ThermoFisher PageRuler prestained protein ladder.Sol: Solubilized TRPM2 after the removal of any insoluble material by a second round of centrifugation.IMAC purified TRPM2 was fully eluted in the four elution steps (Elution 1-4), without passing through to the wash steps (Wash 1-3).

Figure 5 .
Figure 5. CIAS-MS of 10 μM TRPM2 incubated with a compound pool consisting of 100 compounds, including clotrimazole (each at a concentration of 10 μM). 10 μM compound mixture alone (A) with CID activated and (B) with CID off; 10 μM TRPM2 incubated with the compound pool (C) with CID activated and (D) with CID off.