# Development of ultra-miniaturized weak affinity chromatography coupled to mass spectrometry as a high throughput fragment screening method against wild-type and purified membrane proteins embedded in biomimetic membranes

François-Xavier Vidal, Julie Gil, Maud Gregson, Gabrielle Zeder-Lutz, Maria Hideux, Jérôme Lemoine, Isabelle Krimm, Renaud Wagner, Vincent Dugas, Claire Demesmay

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François-Xavier Vidal, Julie Gil, Maud Gregson, Gabrielle Zeder-Lutz, Maria Hideux, et al. Development of ultra-miniaturized weak affinity chromatography coupled to mass spectrometry as a high throughput fragment screening method against wild-type and purified membrane proteins embedded in biomimetic membranes. Analytica Chimica Acta, 2025, 1353, pp.343950. 10.1016/j.aca.2025.343950. hal-05285292

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# Development of ultra-miniaturized weak affinity chromatography coupled to mass spectrometry as a high throughput fragment screening method against wild-type and purified membrane proteins embedded in biomimetic membranes

François-Xavier Vidal<fnref n="1" />, Julie Gil<fnref n="1" />, Maud Gregson<fnref n="1" />, Gabrielle Zeder-Lutz<fnref n="2" />, Maria Hideux<fnref n="3" />, Jérôme Lemoine<fnref n="1" />, Isabelle Krimm<fnref n="4" />, Renaud Wagner<fnref n="2" />, Vincent Dugas<fnref n="1" /> and Claire Demesmay<fnref n="1" />,*

## Abstract

**Background:** Membrane proteins, which make up approximately 30% of the proteome, are important drug targets but present many challenges in drug discovery, including limited production rates, low final yields of pure and functionally folded proteins, and instability in aqueous media. The problems encountered with membrane proteins are even more critical in the Fragment Based Drug Discovery, where the discovery of potential drug candidates is hampered by the limited availability of efficient methods for rapid screening of weak fragment-protein interactions.

**Results:** In this work, we propose the coupling of miniaturized weak affinity chromatography with mass spectrometry (nano-WAC-MS) as an innovative strategy for the rapid screening of fragments capable of weak binding to a selected membrane protein. An integral membrane protein (AA₂AR) was incorporated into biotinylated nanodiscs, which were subsequently immobilized on a miniaturized monolithic streptavidin column (75µm i.d., 300 nL volume). The coupling of these miniaturized affinity columns (each consuming less than 1µg of protein) to mass spectrometry (MRM mode) has been optimized to maximize the low affinity range and increase throughput so that 150 fragments can be injected in a single analysis, with a DMSO content as high as 10%, with no influence on the affinity. Hits are identified by comparing their retention with that measured on control columns prepared with empty nanodiscs.

**Significance:** The results of this screening are compared with those obtained by NMR and newly identified hits are confirmed by either competition experiments or frontal affinity experiments. We show that this nanodisc-based strategy, which provides a stable and native-like lipid environment for the protein (columns can be used for several days), should also work with other membrane proteins embedded in nanodiscs.

**Keywords:** Weak Affinity Chromatography; fragment-based drug discovery; monolithic capillary column; nanoliquid chromatography; membrane protein; mass spectrometry

<footnote n="1">¹ Universite Claude Bernard Lyon1, ISA, UMR5280, CNRS, 5 rue de la Doua, 69100 Villeurbanne, FRANCE. France.</footnote>

<footnote n="2">² Plateforme IMPReSs, CNRS UAR3286, Plateforme de chimie biologique intégrative de Strasbourg, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.</footnote>

<footnote n="3">³ Institut de recherche et développement Servier Paris-Saclay-22, route 128, 91190 Gif sur Yvette - France.</footnote>

<footnote n="4">⁴ Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon L 69373, France</footnote>

<footnote>* Correspondence: Demesmay@univ-lyon1.fr</footnote>
# 1- Introduction

Alongside traditional HTS approaches, Fragment Based Drug Discovery (FBDD), introduced in the 1990s, has opened up new avenues in drug discovery. FBDD has established itself as a viable approach to the discovery of new therapeutic molecules, with seven drugs approved by the FDA: Ascinimib, Pexidartinib, Capivasertib, Erdafitnib, Sotorasib, Vemurafenib and Venetoclax. FBDD is based on a structure-guided approach: a small-molecule library (low-MW molecules <300 Da) also called "fragment library" is used during the initial screening process against a protein target, to identify binding fragments ("hits") with a potential for subsequent therapeutic development. Due to their inherent small size, binding fragments present only a weak affinity for the target (in the mM-µM range), and these fragments have then to be converted (through iterations guided by structural knowledge of the site of binding) to high-affinity ligands ("drug-like") by linking them together (or bridging or growing). Numerous reviews have described the many benefits and potential of this approach in drug discovery. Screening methods to identify such weakly interacting fragments, are at the heart of FBDD. To date, numerous biophysical techniques (NMR, surface plasmon resonance (SPR), IsoThermalCalorimetry (ITC), Thermophoresis, Thermal Shift Assays (TSA), X-ray Crystallography, affinity selection Mass Spectrometry (ASMS))[1-3] are available or under development for fragment screening mainly against water soluble proteins.

The challenges associated with membrane proteins (MP) in the context of fragment screening relate to different issues that are connected to their intrinsic nature: production rates of MPs are often limited, their extraction and purification necessitate the use of specific surfactant agents, final yields of pure and functionally folded proteins are usually low and their instability in aqueous media is a notorious concern[4,5]. While a number of recent developments now allow to overcome several many of these challenges[6], some of them still prevent the investigation of MPs in solution for fragment screening purposes, such as low amounts of pure and functional material, as well as instability issues. Because of these limitations, the fragment screening assays that have been tentatively developed for MPs mainly rely on proteins that have been artificially modified through mutations or truncations in order to increase their stability in aqueous solution [7]. Besides the fact that these strategies are demanding and time consuming, such stabilizing mutations introduce physical constraints that may influence the structural and dynamic properties of the proteins, thereby questioning the relevance of interacting compounds that could be selected against them.

The present work reports the development of a relevant and efficient screening technology adapted to the specificity of MP manipulation in aqueous solution ideally working with low amounts of unmodified proteins (unbiased conformational state) in a rapid screening format. The chosen technology is based on weak affinity chromatography coupled to mass spectrometry, which has already been proposed as a complementary screening method in FBDD [8]. To date only one article reports its application to a membrane protein [9] (human aquaporin-1) chemically immobilized on silica stationary phases after its in stabilization in planar lipid bilayer disks (lipodisks). The use of these lipodisks-functionalized columns coupled to mass spectrometry allowed screening of 10 fragments/injection for a total screening of 200 fragments in 20 analyses (low-throughput screening in full scan mode). Although this article has demonstrated the interest of WAC-MS for the screening of fragments on membrane proteins, it seems necessary to develop it further (more oriented and homogeneous protein immobilization, higher throughput) in the hope of making it a reference method in this field. This has led our group to work on successive developments aimed at reducing the consumption of MP [10], increasing the surface density of active proteins [11], limiting the non-specific retention of fragments [12,13] and extending the affinity range to low affinities [11]. We designed nano-WAC columns implementing hydrophilic monolithic stationary phase in 75 µm i.d. capillaries for the oriented immobilization of wild-type, purified MP stabilized in nanodiscs as biomimetic membranes[13]. Lipid nanodiscs, formed by the cooperative assembly of phospholipids and an amphiphatic protein (membrane scaffold protein), help to overcome the instability of membrane proteins in aqueous solutions by preventing exposure to the aqueous environment. The lipid molecules
self-assemble into a bilayer structure in the aqueous phase, and the membrane scaffold protein acts
as a stabilizer by wrapping around the edges of the lipid bilayer to prevent its disassembly. The bilayer
structure simulates the environment of the cell membrane, allowing biological macromolecules such
as membrane proteins to maintain activity and function in a near-natural state. In addition, when NDs
are immobilized through the biotinylated MSP belt, both intracellular and extracellular surfaces of the
MP are accessible to any interacting partners. As only the MSP wrapping the nanodisc is biotinylated
(the target protein itself remains fully native), the approach is intended to be generic and directly
applicable to any membrane protein reconstituted in nanodiscs. This immobilization method
drastically reduces MP consumption to immobilized MP only. As a result, a sub-microgram amount of
MP is sufficient to produce a miniaturized column with a high surface protein density (i.e
approximately 30 pmol for a 8-cm length column). These improvements allow the detection of low
affinities down to a few hundred µM while reducing protein consumption by a factor of 3-4 compared
to the previous study [9]. The total amount of protein consumed is lower due to miniaturization, but
the volume density (B<sub>act</sub>/V<sub>m</sub>) is higher, so the range of detectable affinities is wider (i.e. it is easier to
detect low affinity fragments).

Here, we describe the implementation of the coupling of these nano-WAC columns to mass spectrometry (MS) in the context of fragment screening to reach a high throughput screening. Experimental coupling conditions (flow rates, make-up, DMSO content) were optimized to (i) obtain stable signal throughout the chromatogram timeframe, (ii) detect fragments at low concentration in purely aqueous (buffered) mobile phase in stable state (iii) from a sample mixture of the largest number of fragments. These optimized conditions were used to realize a fragment screening using the adenosine A2A receptor (AA2AR), a prototypical class A GPCR receptor. In addition to its biological relevance [14–17], this model benefits from a large repertoire of known ligands [18,19] which is useful for the development and validation of new methods. The screening methodology used is based on the comparison of retention times between the AA2AR affinity column carrying the receptor in its nanodisc and a control column carrying nanodiscs without the receptor inside (empty nanodisc). The screening was performed using a set of 121 fragments that had been used to screen the same AA2AR GPCR model receptor by NMR and prepared in detergent. The conditions for the retention time shift threshold to be applied between the affinity and control columns were evaluated in order to define hit detection method.

2- Material and methods

2.1 - Reagents and buffers

(3-methacryloxypropyl)-trimethoxysilane (γ-MAPS); glycidyl methacrylate (GMA); acrylamide; N,N'-Methylenebis(acrylamide) (MBA); dodecanol; 1,4-butanediol; dimethyl sulfoxide (DMSO); sodium periodate; lithium hydroxide; dipotassium hydrogen phosphate (K₂HPO₄); o-phosphoric acid; ammonium acetate, sodium cyanoborohydride; triethylamine (TEA); azobis(isobutyronitrile) (AIBN); Streptavidin (from Streptomyces avidini, affinity purified, ≥13 U.mg⁻¹ of protein); and fragments' library were purchased from Sigma-Aldrich (L'Isle d'Abeau Chesne, France). All aqueous solutions were prepared using >18 MΩ of deionized water. Phosphate buffer 67 mM was prepared by dissolving 1.17 g of K₂HPO₄ in 100 mL of ultrapure water, and the pH was adjusted to 7.4 with phosphoric acid. Acetate solution was prepared by dissolving 1.54 g (ammonium acetate 20mM) or 12.3 g (ammonium acetate 160mM) in 100mL of ultrapure water, and the pH was adjusted to 7.4 with ammonia.

2.2 Preparation of affinity monolithic capillary columns

2.2.1 Capillary columns activation

Commercially available fused-silica capillaries (75 µm i.d., 375 µm o.d.) coated with polyimide (TSP) were obtained from Cluzeau Info Labo (Sainte-Foy-La-Grande, France). 15-cm length capillaries were activated by flushing a methanol/water mixture (95/5, v/v) containing 5% (v/v) of γ-MAPS and 2.5%
(v/v) of TEA for 1 hour at a pressure of 7 bar with a pressurization device (Nanobaume™ supplied by Western Fluids Engineering, Wildomar, USA). Capillaries were then rinsed with methanol for 15 minutes at 7 bar and dried at room temperature under a nitrogen flow before use.

### 2.2.2 Synthesis of poly(GMA-co-MBA) monolith

The poly(GMA-co-MBA) monoliths [12] were prepared by mixing 1480 mg of 1,4-butanediol, 1850 mg of dodecanol, 320 mg of MBA, and 480 mg of GMA in 3330 mg of DMSO for 1 h. A total of 8 mg of AIBN was added, and the final mixture was sonicated for 15 min. The activated capillary was then filled with the polymerization mixture under 1 bar of N₂ pressure and the ends of the capillary were sealed. The polymerization reaction was performed in a water bath at 57°C for 18 h. After polymerization, the monoliths were rinsed with methanol for 1 h and kept wet until use.

### 2.2.3 Preparation of generic-streptavidin poly(GMA-co-MBA) monolithic capillary columns

Poly(GMA-co-MBA) monoliths were hydrolyzed in water at 80°C for 18 hours. Monoliths were then flushed with a 0.12 M NaIO₄ solution at pH 5.5 for 1 hour at 7 bar to convert theiol into aldehyde groups. Streptavidin was covalently grafted through their amine groups by percolating a 1 mg.mL⁻¹ streptavidin solution prepared a 67 mM phosphate buffer (pH 6) containing 4 mg.mL⁻¹ NaBH₃CN in for 18 hours at 7 bar at room temperature. After immobilization, the columns were flushed with a sodium borohydride solution (2.5 mg.mL⁻¹ in 67 mM phosphate buffer, pH 8) for 2 hours at 7 bar to reduce residual aldehydes. Finally, the streptavidin-functionalized columns were rinsed with phosphate buffer and stored at 4°C.

### 2.2.4 Preparation of NDs-functionalized monolithic capillary columns (nano-WAC columns)

Control and affinity nanoWAC columns were prepared by percolating NDs with or without the AA₂AR receptor incorporated. Biotinylated-ND solutions (in the micromolar range) were percolated through generic-streptavidin columns. Percolation is monitored by in-situ UV detection (280 nm) and stopped when the NDs solution breakthroughs the column (i.e saturation of the streptavidin binding sites). This setup allows quantification of the total amount of ND captured (Btot).

## 2.3. Preparation of nanodisc samples (NDs)

### 2.3.1 Production and purification of AA₂AR

A recombinant his-tagged human AA₂AR was heterologously expressed in Pichia pastoris and purified as previously described [16]. Briefly, the expressed AA₂AR was extracted from whole membrane fractions using a solubilization buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 0.5% n-Dodecyl-β-D-Maltopyranoside (β-DDM) (w/v), 0.05% Cholesteryl Hemisuccinate (CHS) (w/v), 30 mM imidazole, 1 µM 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), 0.3 mM EDTA, one protease inhibitor tablet) before purification on an IMAC column (1 mL HisTrap HP column, Cytiva, Marlborough, MA, USA) in a buffer containing 50 mM HEPES pH 7.4, 500 mM NaCl, 0.05% β-DDM (w/v), 0.005% CHS (w/v), 100 mM imidazole, and 1 µM DPCPX. The resulting fractions were pooled and loaded onto a HiLoad Superdex 200 Increase 16/600 PG column (Cytiva, Marlborough, MA, USA) in a buffer consisting of 50 mM HEPES pH 7.4, 150 mM NaCl, 0.02% β-DDM (w/v), 0.002% CHS (w/v), and 1 µM DPCPX. Fractions corresponding to the monomeric AA₂AR in detergent were pooled, concentrated to approximately 0.8 mg.mL⁻¹ using a Vivaspin20 30K MWCO device (Sartorius, Bangkok, Thailand), and directly used for reconstitution into lipid nanodiscs.

### 2.3.2 - Production, Purification, and Biotinylation of Membrane Scaffold Protein (MSP)

The membrane scaffold protein MSP1E3D1(-) was expressed and purified from E. coli following the protocol described in [20]. Biotinylation of MSP1E3D1(-) was performed using Thermo Scientific's EZ-Link™ NHS-PEG4-Biotin reagent, as previously reported [10]. The biotinylated protein was further purified on a HiTrap Desalting column (Cytiva) in a storage buffer containing 20 mM Tris-HCl pH 7.4,
100 mM NaCl, and 0.5 mM EDTA. Following snap freezing in liquid nitrogen, the biotinylated MSP1E3D1(-) was stored at -80 °C until use.

### 2.3.3 - Assembly and Purification of Empty NDs and AA₂AR-Loaded NDs

The biotinylated MSP1E3D1(-) was combined with purified lipids at a 1:70 molar ratio. The zwitterionic phospholipids (POPC, Avanti Polar Lipids, Alabaster, Alabama, USA), dissolved at 24 mM, were prepared in a buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, and 48 mM sodium cholate. The MSP: lipid mixture was incubated on ice for 15 minutes before adding the detergent-purified AA₂AR at a 1:10 AA₂AR:MSP1E3D1 (-) molar ratio. This mixture was incubated on ice for an additional 60 minutes.

Nanodisc self-assembly was initiated and enhanced by detergent removal using BioBeads SM-2 (Biorad, Hercules, CA, USA), added at 0.25 g.mL⁻¹ of the reconstitution mixture. After overnight incubation at 4 °C on a tube rotator, the BioBeads were removed by centrifugation. The resulting supernatants were directly injected onto a 1 mL HisTrap HP column (Cytiva, Marlborough, MA, USA) pre-equilibrated in a buffer of 50 mM HEPES pH 7.4, 300 mM NaCl, and 10 mM imidazole.

Flow-through fractions, predominantly containing empty NDs, were collected for further purification via size exclusion chromatography. After extensive washing of the column with the equilibration buffer, AA₂AR-loaded NDs were eluted with 500 mM imidazole and pooled. Both types of samples (empty NDs and AA2AR-loaded NDs) were then subjected to purification on a Superdex 200 Increase 10/300 GL column (Cytiva, Marlborough, MA, USA), pre-equilibrated with a buffer containing 50 mM HEPES pH 7.4 and 150 mM NaCl. Elution was performed at a flow rate of 0.3 mL.min⁻¹.

The collected fractions, corresponding to purified empty NDs (POPC), AA₂AR NDs (POPC) were aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until further use.

## 2.4 Fragment library

All fragments (for chemical information, CAS numbers, and code names see Table S1) were solubilized individually at 110 mM in 100% DMSO (stock solution), except for a few fragments which were solubilized in 100% MeOH. For screening, the fragments were diluted to a final concentration of 100 µM in an aqueous solution of 20 mM ammonium acetate at pH 7.4 (the final DMSO content depends on the size of the mixture, about 11% DMSO for a mixture of 121 fragments).

To identify and optimize the MRM transitions for each fragment, mixtures of 8 fragments at 50 µM each were prepared in an aqueous solution of 20 mM ammonium acetate at pH 7.4 and methanol, 20/80.

## 2.5 Staircase Frontal Affinity Chromatography (FAC)

FAC experiments (for hit confirmation or for the determination of the number of active binding sites Bₐct of streptavidin or AA₂AR) were implemented on the Ultimate 3000 Thermo UHPLC system modified with a 10-port valve equipped with two superloops (5 µL), operating alternately to infuse automatically ligand solutions at increasing concentrations for AA₂AR column qualification (staircase experiments) [21]. In frontal affinity chromatography, UV detection was implemented at 260nm for caffeine as test ligand for AA2A, 342nm for HABA (test ligand for streptavidin binding sites) and 214 and 280 nm for nanodiscs.

## 2.6 Nano-LC-MS Experiments

### 2.6.1 Fragment screening by nanoWAC-mass spectrometry coupling

Fragment screening experiments were carried out on both affinity columns and control columns using the Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific, San Jose, CA, USA)
controlled by Chromeleon software, coupled to a hybrid triple quadrupole ion trap mass spectrometer (QTRAP 6500+, AB Sciex, Foster City, CA, USA) equipped with an OptiFlow™ Turbo V ion source from SCIEX and a 1-10 µL probe to accommodate low flow rates. Fragment elution was monitored by Multiple Reaction Monitoring (MRM) mode. Data acquisition and processing were performed using the SCIEX OS software version 3.3.0.12027.

For selecting and optimizing the MRM transitions, all fragment compounds prepared at a concentration of 50 µM in an 8-fragment mixture were infused directly into the ion source at a flow rate of 1 µL.min⁻¹, in both positive and negative ionization modes, to evaluate the most appropriate mode as well as the most intense transitions. The resolutions of quadrupoles Q1 and Q3 were adjusted to unit resolution. After optimization, the following source parameters were used: spray voltage set to 5.5 kV, a source temperature set to 200°C, curtain gas: 20 psi, GS1 : 60 psi, GS2 : 90 psi. The best transitions and optimal fragmentation values (collision energy and declustering potential) were chosen to avoid overlapping transitions for each fragment; in such cases, a second transition was added for the fragment.

The screening was performed using an injection volume of 16 nL (nano-valve VICI C74NH4-4674) with an internal loop and a flow rate of 200 nL.min⁻¹ at room temperature. Analyses were performed under isocratic conditions using an ammonium acetate (20 mM, pH 7.4 adjusted with ammonia) mobile phase. An additional make-up flow (MeOH at 800 nL.min⁻¹) was added with the loading pump of the nano-LC device and mixed with the chromatographic flow in a zero-dead volume union (UH-750 from IDEX (Cluzeau info labo, France) to stabilize the electrospray. The dead time was assessed by monitoring the DMSO with transition 79.13 Q1 and 63.998 Q3.

### 2.6.2 Data Analysis:

Data analysis was performed using the SCIEX OS software (AB SCIEX), with the Autopeak integration algorithm for peak integration. The peak integration parameters were set as follows: low smoothing with the noise filter enabled, interference resolution set to 50%, peak baseline was neither local nor linear, and a re-centering window of 300 seconds around the expected retention time.

## 3. Results and Discussion

### 3-1 Preparation and characterization of affinity and control nano-columns

Nano-WAC affinity and control columns were synthesized as described in Figure 1. A poly(GMA-co-MBA) monolith was in-situ synthesized in 75-µm i.d. fused silica capillary. This monolith was chosen for its higher hydrophilicity when compared to the previously used gold standard poly(GMA-co-EDMA)[13]. Generic streptavidin functionalized monoliths were prepared by coupling streptavidin to the monolith through the Schiff base method after conversion of glycidyl groups to more reactive aldehyde groups. The generic streptavidin columns were further modified by percolating the biotinylated ND solution with (+AA₂AR) or without (-AA₂AR) the AA₂AR target receptor to prepare affinity and control nano-WAC columns, respectively. As shown in Figure 1, the NDs are immobilized on the monolith by the biotinylated MSP, so that the membrane protein remains fully native (no conformational change) and is fully accessible (intra and extra-cellular sides).

The receptor is thus fully accessible (intra and extracellular sites) NDs with zwitterionic phospholipids (phosphatidylcholine, POPC) were chosen to minimize non-specific interaction due to the assembly[13].
<figure><img src="image_2.png" /><figcaption>Figure 1. Schematic representation of nano-WAC column fabrication from in-capillary poly(GMA-co-MBA) synthesis, streptavidin biofunctionalization to biotinylated nanodiscs (ND-biotin) grafting.</figcaption></figure>

Although the preparation conditions of the nano-WAC columns have been thoroughly investigated, the use of such NDs-functionalized poly(GMA-co-MBA) nano-WAC columns in the context of fragment screening requires prior specific qualification to ensure consistency of retention time measurements. This is achieved by stepwise control. The columns are checked at various stages of manufacture to ensure that the synthesis and grafting steps result in homogeneous columns within and between batches. The polymerization step is controlled by measuring the permeability of the monoliths. The number of streptavidin binding sites on each column is determined by staircase frontal affinity chromatography using (2-(4-hydroxyphenylazo)benzoic acid (HABA)) as test ligand [22]. Indeed, HABA is a natural low affinity ligand for streptavidin. Each streptavidin molecule has four interaction sites that recognize HABA. Frontal chromatography experiments with HABA can be used to quantify the number of streptavidin recognition sites available, i.e. the number of sites capable of subsequently capturing biotin (biotinylated NDs). Targeted permeability and number of streptavidin binding site ranges are (2.5 ± 0.5) x 10<sup>-14</sup> m<sup>-2</sup> and 140 ± 10 pmol/column respectively. Knowing that one streptavidin has 4 recognition sites, we can estimate the amount of streptavidin to be about 35 pmol per column. The percolation of the nanodisc solutions of known concentration is UV-monitored, allowing the determination of the total amount of NDs captured (B<sub>tot</sub> = 30 ± 5 pmol/ column). Finally, the amount of active membrane protein (B<sub>act</sub>) (able to bind ligands) is determined by staircase frontal affinity chromatography (B<sub>act</sub> = 27 ± 5 pmol/ column, n=7 columns) with caffeine as ligand of known Kd (23 µM [18]).

Column stability over time is essential to ensure consistency of results. The stability of the affinity columns was therefore assessed by characterizing the AA<sub>2AR</sub> ability to bind its ligands through affinity measurements with caffeine over time (Figure S1, Supplementary material). The lifetime of the columns depends mainly on the temperature and duration of use or storage, rather than on the operating conditions (flow rate, cumulative volume of mobile phase percolated). Under operating conditions (i.e. at room temperature, with ammonium acetate 20mM pH=7.4 continuously percolated)), the number of protein binding sites decreases steadily over time. This is probably due to the partial denaturation of the protein over time at room temperature. By setting an activity drop threshold of 30%, we have shown that it is possible to work with affinity columns prepared with NDs under screening conditions for at least four days. In-between analysis the column can be stored at 4°C. This decrease in activity leads to a reduction in the retention time shift by affinity of the same order of
magnitude and therefore to an acceptable reduction in the range of detectable affinity over time. For
systematic control, caffeine with a known binding constant to the protein, is systematically added to
the sample mixture, to quantitatively monitor column performances (protein activity).

3.2 Optimization of the NanoWAC-MS coupling

Coupling affinity nano-columns, operating in pure aqueous and buffered mobile phases, to MS
detection requires optimization of experimental parameters to ensure a level of stability and detection
threshold suitable for use in fragment screening. MS detection has been implemented in MRM mode
to increase detection specificity and sensitivity and to allow the injection of complex fragment mixtures
(mixtures containing up to 150 molecules). One MRM transition was selected for each fragment (See
supplementary material Table S1). Considering the maximum number of MRM transitions that can be
entered into a single acquisition method with the acquisition software (SCIEX OS software version
3.3.0.12027), the maximum mixture size is 150 fragments. To scan 1500 fragments, 10 acquisition
methods (with different MRM transitions) are required and can be run automatically in a programmed
sample list.

3.2.1 Optimization of the coupling conditions

Addition of a make-up flow : Affinity analysis were performed in zonal mode in the presence of a 100% aqueous mobile phase (20 mM ammonium acetate at pH=7,4) at a flow rate of 200 nL.min⁻¹. MS coupling in pure nano-spray mode is not possible due to the common problems of nanospray emitter clogging and spray instability in buffered aqueous media. Therefore, coupling was performed using a micro-ESI source (OptiFlowTM Turbo V ion source from SCIEX). This ionization source works at optimum operating flow rate between 1 and 10 µL.min⁻¹. A methanol make-up flow is added to the column outlet (via a zero-dead-volume fitting) to stabilize the spray and promote ionization of the compounds. The influence of the liquid flow rate was studied to obtain the best compromise between robustness and sensitivity. The addition of methanol to the 200 nL.min⁻¹ mobile phase flow rate at the column outlet promotes the ionization of the solutes and the robustness of the spray but is accompanied by a dilution of the sample. The minimum additional liquid flow rate is 800 nL.min⁻¹ to reach the recommended operating range of the source (1 to 10 µL.min⁻¹). As illustrated Figure S2 (for a selected set of 12 fragments including cations, neutral and anions of various hydrophobicity), increasing the additional liquid flow rate up to 2 µL.min⁻¹ resulted in an average signal intensity reduction of 70 % for all compounds, with a maximum of up to 90% for some of them. This loss of signal, which was greater than the increase in dilution effect, is attributed to a decrease in ionization efficiency at high flow rates. In the selected range of make-up flowrate, no difference of spray stability was observed. Thus, a make-up flow rate of 800 nL.min⁻¹ was chosen for further work.

3.2.2 Influence of DMSO in the fragment mixture on the sensitivity

In fragment libraries, fragment stock solutions are usually prepared in 100% DMSO. When mixtures of
fragments are prepared, the final percentage of DMSO in each mixture depends on both the number
of fragments per mixture and the concentration of each fragment in the mixture. In mass
spectrometry, DMSO is known to interfere with the ionization of solutes by producing very intense
adducts that remain in the source. [23] The presence of DMSO in the source was monitored by plotting
extracted-ion chromatogram at m/z = 79.5, which is characteristic of the [DMSO+H]⁺ adduct, for
various DMSO contents in the injection solvent, ranging between 2 and 10% (see Figure S3). Even with
a very low injection volume of 16 nL, DMSO is detected in the source throughout the chromatogram.
The influence of the percentage of DMSO on the MS signal intensity for a set of 40 fragments is shown
in Figure 2. For this set of fragments, with retention factors between 0 to 11 (representative of the
whole range of retention factors for the entire library), increasing the percentage of DMSO in the
injection buffer from 0.1 to 5% resulted in a 50-60% signal reduction. From 5 to 10% DMSO, there was
no significant effect of increasing DMSO content on the signal.

<figure><img src="image_3.png" /></figure>

Figure 2. Graphic comparing the signal intensity of a mix of 40 fragments for different DMSO contents in the fragment mixture (v/v %); 0.1% in blue, 5% in orange, and 10% in gray. Fragments are sorted by charge state and increasing logD values. Injection of 16 nL of the mix of fragments at 100 µM each on an 8-cm length empty NDs column. MS detection was conducted in MRM mode by using positive or negative mode (*). Separation was done using a 20 mM ammonium acetate (pH 7.4) mobile phase at 0.2 µL.min⁻¹ and MeOH make-up flow rate of 0.8 µL.min⁻¹.

Under these analytical conditions and with an injection volume of 16 nL, all of the fragments are
detectable at a concentration of 50 µM. Considering fragment stock solutions at 100 mM prepared in
pure DMSO, it is possible to prepare mixtures of up to 200 compounds at 50 µM of each fragment with
a final DMSO content of no more than 10%. DMSO is therefore not a strong limitation in terms of
sensitivity to the size of the mixtures.

3.2.3 Effect of DMSO on protein binding affinity

DMSO, as an organic solvent, may affect the protein-fragment binding affinity [23]. While DMSO has a
significant effect on the affinity of the fragments for the protein in frontal affinity chromatography, it
has no effect in zonal mode. This is illustrated in Figure 3, which shows that the retention factor of
caffeine measured in zonal mode is not affected by the presence of 10% DMSO in the injection solvent,
whereas the decrease in caffeine breakthrough time with increasing the DMSO in frontal mode reflects
a decrease in affinity (lower amount of ligand captured in the presence of DMSO). Indeed, as fragments
interact with the column in zonal mode (whether specifically or non-specifically), they are slowed down
relative to the solvent (that is eluted at the dead volume) and therefore separated from the injection
solvent plug.
<figure><img src="image_4.png" /><figcaption>Figure 3. Chromatograms (reduced retention time on the x-axis) showing the retention of caffeine by affinity chromatography in zonal mode (figure 3, A left) and frontal mode (figure 3, B right) for different DMSO contents (0, 2 and 10 %) in the sample solution. Injection of 200 µM caffeine solutions (A) and infusion of a 25 µM caffeine solution (B) on 8.5-cm length column functionalized with AA₂AR NDs using a 20 mM ammonium acetate (pH 7.4). UV detection at 260 nm.</figcaption></figure>

### 3.3 Fragment screening by NanoWAC-MS

The 121 fragments selected for the screening are fragments (See supplementary material Table S1 for CAS numbers, LogD and Log P values) that have already been screened by NMR [24] (STD experiments with the receptor solubilized in micelles). These 121 fragments were initially randomly selected from an FBDD library. Since these fragments may interact non-specifically with the material inside the capillary column (monolith, streptavidin, constituents of the nanodisc), it is necessary to measure retention factors on control columns ($k_{ns}$) and compare them with those measured on affinity columns ($k_{ns} + k_{affinity}$) by calculating the $\Delta k$ value which represents the affinity-related retention factor ($k_{affinity}$). In affinity chromatography, the affinity-related retention factor depends on the amount of active sites and the concentration of the ligand according to the following relationship:

$$k_{affinity} = \frac{1}{K_{D} + [L]} \times \frac{B_{act}}{V_{m}}$$

To maximize affinity-related retention, it is best to work in conditions where the equilibrium ligand concentration [L] is negligible compared to the $K_d$ value (linear region of the adsorption isotherm). However, due to the chromatographic dilution effect, an injection concentration of the order of magnitude of the $K_d$ allows one to be in this linear zone of the isotherm. For example, it has been shown experimentally that the retention factor of caffeine ($K_d$ = 25 µM) measured on an affinity column (NDs+AA₂AR) is independent of the concentration for injection concentrations up to 100 µM. For fragments of lower affinity, this condition is even more likely to be met.

Under these conditions, the measurement of $\Delta k$, i.e. the $k_{affinity}$, allows a direct estimation of the $K_d$ of the interaction, provided that the ratio $B_{act}/V_m$ has been previously determined.

#### 3.3.1 Control column considerations

To discriminate a specific interaction from a non-specific one, it is essential to compare the retention factors of the fragments on the affinity column with a reference value obtained on a control column. The control column should allow the best estimation of the non-specific interactions due to the support and the assembly structure (in the absence of the receptor). In the case of ND-encapsulated GPCR affinity columns, two types of control columns can be considered: control columns grafted with the nanodiscs without the receptor (NDs-AA₂AR) (called “empty nanodiscs”) [25], or control columns
in which the orthosteric site of the AA<sub>2A</sub>R is blocked with a known ligand. In the latter case, all experiments are performed on the same column. The first screening is performed on an affinity column with the fully accessible protein receptor, followed by a second screening on the same column after saturation of the receptor with a strong ligand that blocks the accessibility of fragments to orthosteric sites. This two-step method is designed to measure specific and non-specific interactions, respectively. One of the key features of this method is that it does not need to account for inter-column variability but it is not universal. Firstly, the method relies on the knowledge of a strong ligand which precludes screening against orphan receptors. It also does not allow the identification of allosteric ligands. Secondly, it should be noted that non-specific interactions must be measured for each screening, *i.e.* for each target protein. Finally, the nature of the ligand (agonist, antagonist, etc.) relative to the GPCR influences the populations of different conformational states of the protein between the basal and active forms which may present different interaction surfaces and bias the measurement of non-specific interactions.

We have therefore chosen the first option, where a column with empty NDs is used as a control column. This method is more universal. A first screening is performed only once to measure non-specific interactions, regardless of the receptor being studied. This second approach is applicable to any receptor, including orphan receptors for which no ligand is known. In addition, this method is not limited to detecting interactions with the orthosteric site, but remains open to the discovery of allosteric ligands. The main disadvantage is that two different columns are used to measure non-specific and specific interactions, and the variability between columns must be taken into account to define the criterion for deciding which fragment is a hit, i.e., which minimum Δk value is significant.

### 3.3.2 Choice of hit identification criterion

Screening is based on the comparison of retention factors between a control column and the affinity column. The retention factor shifts Δk is the difference between the retention factor measured on the affinity column and the control column.

Defining a threshold above which the shift in retention factor can be attributed to a specific interaction is therefore a key element in the development of the nano-WAC-ESI-MS fragment screening method. The choice of the hit identification criterion depends on the variability of the retention factors on the control column (retention factors for non-specific interactions). The greater this variability, the more stringent the hit identification criterion must be.

The characterization of the variability of the retention factors measured on 3 different control columns with empty NDs (retention due to non-specific interactions) was performed by measuring the standard deviations and associated relative standard deviations for 12 fragments with retention factors between 0.5 and 11 (Figure S4). The CVs depend on the compounds and vary from 2% for the weakest to a maximum of 13%. We note a strong overall variation for the poorly retained compounds which seems logical knowing that a small variation in the retention time will lead to a greater variability for low retention factors than for high ones.

Therefore, a single hit identification criterion that is identical over the entire non-specific retention range is not relevant. This observation implies that threshold conditions for the detection of a specific interaction (affinity) should be defined as a function of the non-specific retention factor. Considering that the standard deviations on small non-specific retention factors (k<3) are all less than 0.1, a Δk value > 0.5 was set as a fixed threshold for attributing the retention factor shift to affinity. For the most
non-specifically retained compounds (k>3), a criterion of Δk > 15% between the affinity and the control column was used to attribute retention shift to affinity. With these criteria, fragments with K<sub>d</sub> values less than 200 µM can be identified.

### 3.3.3 Application of nanoWAC-MS to fragment screening on AA<sub>2AR</sub>

For the fragment screening, a 16 nL volume of a mixture containing the 121 fragments at a concentration of 100 µM each and a final DMSO content of approximately 10 % was injected onto the nano-WAC columns. Analyses were performed at a flow rate of 200 nL.min<sup>-1</sup> and took about an half an hour. All fragments were monitored in MRM mode with one transition per fragment allowing a chromatogram to be extracted for each fragment. Figure 4 presents a sampling illustrating the different cases observed (hits and non-hits with different retention ranges).

<figure><img src="image_5.png" /></figure>

Figure 4. Extracted ion chromatograms of 10 fragments on affinity (blue line) and control columns (black line). EICs of non-ligands (left column) and potential identified ligands with their estimated K<sub>d</sub> (right column). Detection in MRM (Q1/Q3 ; [M+H]) : Non-ligand F1 : 108/91 ; F376 : 122/107 ; HABA : 243/141 ; Mol-20 : 417/215 ; Ligands : caffeine : 195/138 ; F271 : 199/171 ; F411 : 231/204 ; F462 : 164/120 ; F468 : 149/134 ; F725 : 136/119.

Table S2 (Supplementary Material) summarizes all the retention factors measured on the control and affinity columns and the associated Δk values. The K<sub>d</sub> estimate is given for the compounds that meet the selection criteria. Of the 121 fragments, 33 fragments were identified as hits with K<sub>d</sub> < 200 µM (Table 1).
<table><caption>Table 1: Hits identified by nano-WAC-MS for the AA₂AR. Estimated Kd values are calculated for a Bact/Vm = 90 pmol.µL⁻¹. Comparison with the hits identified with STD experiments in NMR [26] and results of hit confirmation for given fragments.</caption><thead><tr><th>Fragment</th><th>Estimated Kd</th><th>Hit RMN</th><th>Hit Confirmation</th><th>Fragment</th><th>Estimated Kd</th><th>Hit RMN</th><th>Hit Confirmation</th></tr></thead><tbody><tr><td>Adenosin</td><td>10</td><td>NMR</td><td>-</td><td>F451</td><td></td><td>NMR</td><td>-</td></tr><tr><td>caffeine</td><td>19</td><td>NMR</td><td>-</td><td>F462</td><td>64</td><td>NMR</td><td>(C)</td></tr><tr><td>F117</td><td>46</td><td></td><td>(C)</td><td>F468</td><td>141</td><td>NMR</td><td>-</td></tr><tr><td>F134</td><td>118</td><td></td><td>(C)</td><td>F469</td><td>161</td><td>NMR</td><td>-</td></tr><tr><td>F142</td><td>125</td><td></td><td>-</td><td>F475</td><td></td><td>NMR</td><td>-</td></tr><tr><td>F203</td><td>47</td><td></td><td>(C)</td><td>F479</td><td>35</td><td></td><td>(S)</td></tr><tr><td>F234</td><td>169</td><td>NMR</td><td>-</td><td>F492</td><td>35</td><td></td><td>(C)</td></tr><tr><td>F270</td><td>44</td><td>NMR</td><td>-</td><td>F503</td><td>116</td><td></td><td>(NC)</td></tr><tr><td>F271</td><td>21</td><td></td><td>(C)</td><td>F504</td><td>37</td><td></td><td>-</td></tr><tr><td>F280</td><td>32</td><td></td><td>-</td><td>F513</td><td>75</td><td></td><td>-</td></tr><tr><td>F286</td><td>86</td><td></td><td>-</td><td>F534</td><td>47</td><td>NMR</td><td>-</td></tr><tr><td>F298</td><td>98</td><td></td><td>(NC)</td><td>F552</td><td>167</td><td>NMR</td><td>-</td></tr><tr><td>F335</td><td>94</td><td></td><td>-</td><td>F62</td><td></td><td>NMR</td><td>-</td></tr><tr><td>F337</td><td></td><td>NMR</td><td>-</td><td>F725</td><td>145</td><td></td><td>(C)</td></tr><tr><td>F359</td><td></td><td>NMR</td><td>-</td><td>F74</td><td>27</td><td></td><td>(NC)</td></tr><tr><td>F364</td><td></td><td>NMR</td><td>-</td><td>F791</td><td>40</td><td></td><td>-</td></tr><tr><td>F365</td><td></td><td>NMR</td><td>-</td><td>F796</td><td>34</td><td></td><td>-</td></tr><tr><td>F411</td><td>42</td><td>NMR</td><td>(C)</td><td>F85</td><td>104</td><td></td><td>(NC)</td></tr><tr><td>F427</td><td>113</td><td>NMR</td><td>-</td><td>F95</td><td>177</td><td></td><td>-</td></tr><tr><td>F433</td><td></td><td>NMR</td><td>-</td><td>F97</td><td>151</td><td></td><td>-</td></tr><tr><td>F450</td><td></td><td>NMR</td><td>-</td><td></td><td></td><td></td><td></td></tr></tbody></table>

- = Not evaluated ; (C) = confirmed by competition ; (NC) = Not confirmed by competition ; (S) = confirmed by dose-response experiment in staircase mode

The results of this screening were compared with those obtained with the same set of fragments in STD NMR[26] with membrane proteins in micelles (Figure 5).

<figure><img src="image_6.png" /><figcaption>Figure 5. Venn diagram comparing the number of identified fragments by NMR (STD experiments in micelles) and nanoWAC-MS.</figcaption></figure>
There are 12 hits common to both methods. However, both techniques have their own specificities which means that some fragments are only identified as hits for only one method. NMR is a reference method for soluble protein fragment screening but is not fully suitable for integral membrane proteins. This method has the advantage of high sensitivity allowing the detection of affinities down to mM but is hardly compatible with the use of biomimetic membranes (such as nanodiscs) [27,28] which guarantee the structural flexibility of membrane proteins outside their native membrane. The NMR samples contained AA₂AR solubilized in micelles, while nano-WAC-MS experiments were conducted with the receptor embedded in nanodiscs. As a result, each technique could introduce different non-specific interactions. Generally, NMR is more likely to detect weaker affinities compared to nano-WAC-MS. However, fragments lacking aromatic structures may be harder to detect with NMR due to overlap from aliphatic and buffer signals. It can be assumed that some hits detected by NMR are outside the affinity range of the nanoWAC-MS method developed here. Conversely, some compounds identified by WAC are not identified by NMR. This observation highlights the orthogonality of the techniques, where each method provides complementary information, thereby enhancing the coverage and robustness of hit identification [29]. It is not an issue as several biophysical methods are usually used in combination to identify hits. [30,31].

### 3.3.4 Hit confirmation

In affinity chromatography, a more specific characterization of the interaction can be performed by frontal affinity chromatography using either dose-response or competition experiments as second-line methods. For hits with weak non-specific interactions i.e. low retention factor on the control column, dose-response experiments can be performed to give a more accurate value of the Kd value. For hits with strong non-specific interactions i.e. high retention factors on the control column, competition experiments are preferred to confirm the interaction (displacement of an indicator i.e. a known ligand of the receptor with fragment solutions of increasing concentrations). Several hits have been confirmed with such experiments. An example of both experiments is illustrated Figure 6 for F479 (staircase frontal affinity chromatography leading to the following Kd value K<sub>d</sub> = 34 ± 3 µM) and F411 (competition experiments with caffeine as indicator).

<figure><img src="image_7.png" /><figcaption>Figure 6. Experiment of hit confirmation. Hit confirmation of F411 by competition with caffeine at 25µM (A on the left). Dose response confirmation of F479 by frontal mode (B on the right). Affinity chromatography experiments using increasing on AA₂AR affinity nanoWAC column. Column dimension: l=8.5 cm; i.d = 75 µm; UV detection at 260 nm.</figcaption></figure>
## 4. Conclusion

In this work, we demonstrate that the coupling of miniaturized weak affinity chromatography with mass spectrometry (nano-WAC-MS) is an efficient strategy for the rapid screening of fragments capable of weakly binding to a selected membrane protein. An integral membrane protein (AA₂AR) was incorporated into biotinylated nanodiscs, which were subsequently immobilized on a miniaturized monolithic streptavidin column (75µm i.d., 300 nL volume). A control quality of synthesized columns is proposed at each step of their fabrication to guarantee the reproducibility of the synthesis. The coupling of these miniaturized affinity columns (each consuming less than 1µg of protein) to mass spectrometry (MRM mode) has been optimized to maximize the sensitivity and the detection throughput so that 150 fragments can be injected in a single analysis, with a DMSO content as high as 10%, with no influence on the affinity or stability of the receptor.

This multiplexing degree of 150 (number of fragments assessed in a single analysis) is unrivalled: only a few fragments can be studied simultaneously by NMR or crystallography, whereas SPR and TSA can only assess fragments one at a time. Screening larger mixtures with STD-NMR can be challenging.

Thanks to multiplexing and miniaturization, protein consumption is extremely low (1µg/column i.e per screening), which is a huge advantage over other biophysical techniques (NMR, TSA, SPR), especially for membrane proteins that are difficult to express and purify. For example, X-ray crystallography-based fragment screening for membrane proteins requires reproducible, high-quality crystals in large quantities, which is highly challenging. The high degree of multiplexing (due to the separation power of chromatography combined with the detection specificity of mass spectrometry) leads to a screening throughput of about 3500 fragments/day, which is much higher than the throughput in STD-RMN (screening of 1000 fragments in cocktails of 5-10 fragments typically takes 24 hours to several days). Moreover, there are no limitations related to fragment solubility, as in NMR or SPR, where low-affinity fragments must be used at a higher concentration the lower the affinity. In WAC, however, all fragments must be studied at concentrations below 0.1 Kd.

33 hits have been identified by comparing their retention with the one measured on control columns prepared with empty nanodiscs. The results of this screening are compared with those obtained by NMR (STD experiments with the receptor stabilized in micelles) and newly identified hits are confirmed by either competition experiments or frontal affinity experiments. The approach may be indeed generalized to any membrane protein, provided they can be prepared in NDs. This ND technology has been widely applied to a large spectrum of membrane proteins, including multi-subunit and other complex proteins (see the following very recent review [32]), and the various references and examples therein. This minimal membrane-mimicking particles has been shown particularly well suited to highly dynamic membrane proteins such as GPCRs, which is the case in our study, as these can freely float in a lipidic environment and explore their different conformational states. In addition, our experimental format where the particles are immobilized on the support via the MSP belt and not via the membrane protein doesn't impair this flexibility.

## CRediT authorship contribution statement

**François-Xavier Vidal**: methodology, investigation, validation, and formal analysis. **Julie Gil**: investigation validation, and formal analysis. **Maud Gregson**: investigation. **Gabrielle Zeder-Lutz**: investigation. **Maria Hideux**: supervision. **Jérôme Lemoine**: Methodology. **Isabelle Krimm**: Methodology, resources. **Renaud Wagner**: investigation, resources, supervision, and writing—review and editing. **Vincent Dugas**: conceptualization, methodology, validation, supervision, writing—review
and editing, and project administration. **Claire Demesmay**: Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge the support of the French Agence Nationale de la Recherche (ANR) under grant ANR-21-CE29-0012 (project NanoWAC).

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