Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Abstract Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.

Abstract: Mass spectrometry (MS) applications for intact protein complexes typically require electrospray( ES) ionization and have not been achieved via direct desorption from surfaces.D esorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules.Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of an ative DESI platform. By establishing conditions that preserve non-covalent interactions we exploit the surface to capture ar apid turnover enzyme-substrate complex and to optimise detergents for membrane protein study.W ed emonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from am ix of related agonists for specific binding to aG PCR. Overall therefore we introduce this native DESI platform with the potential for high-throughput ligand screening of some of the most challenging drug targets including GPCRs.
A range of applications,i ncluding 2D imaging of small molecules and metabolites released from tissue cross-sections, has become possible with the introduction of powerful DESI approaches when coupled with MS. [1,2] Thep rimary goal of DESI applications has been to focus on the small molecules released, for example in the real-time detection of tumour tissue during surgical procedures. [3,4] While DESI has also been adapted to study large biomolecules,v ia the mixing of ES droplets and solution in ap rocess known as liquid DESI, [5,6] it has not yet been applied to proteins deposited on surfaces and desorbed in solutions that retain their native state interactions.D espite considerable progress in applications of non-denaturing or native MS (nMS) of soluble [7][8][9] and membrane embedded proteins [10] the possibility of effectively "lifting" intact complexes from surfaces is desirable since many high throughput technologies then become accessible. [11] Moreover the lipid distribution in natural membranes is essentially planar and asymmetric with varying spatial and temporal arrangements in the vicinity of embedded protein complexes. [12] Thel ipid distribution and the desire for as urface technology that is also able to analyse membrane proteins motivated us to develop amodified DESI approach capable of releasing folded protein molecules from planar surfaces and to construct an interface that we could couple to ah igh-resolution Orbitrap MS optimised for high mass transmission [13] of membrane proteins. [14] We demonstrate the potential of this methodology in three ways 1) by capturing transient protein substrate products 2) by screening for optimal solution/purification conditions using picoMoles of membrane and soluble proteins and 3) by carrying out ligand binding experiments on planar surfaces.
We modified the design of the original DESI set-up [1] and with our custom-built ion source,ESdevice and sample stage coupled our interface to an Orbitrap Q-Exactive ( Figure 1a and Supporting Information Figure S1). We found that signal intensity was significantly improved if the length of the sample transfer tube,u sed in conventional DESI set-ups, [15] was minimised and the stage was located directly under the inlet of our ion source.W eo ptimised signal intensity using hen egg-white lysozyme and found that the spectra recorded under these native DESI conditions are similar to those from typical nanoflow ES capillaries,implying that the folded state of the protein is maintained (Figure 1b). If the native fold is maintained then deposited lysozyme should be able to carry out enzymatic functions.A ccordingly with N-acetyl-glucosamine (NAG)s ubstrate added to the desorption spray and directed at lysozyme deposited on the stage we observed additional peaks assigned to binding of intact NAG-5 to lysozyme ( Figure 1c). Ther apid turnover of this substrate precludes its observation in solution-based ES [16] but since substrate binding takes place during rapid desorption, analogous to reactive DESI experiments reported for small molecules, [17] the transient bound state can be captured using this native DESI approach.
To investigate application to larger protein assemblies we chose complexes with ar ange of oligomeric states:b ovine serum albumin, tetrameric alcohol dehydrogenase (ADH) and the GroEL 14-mer .W ew ere able to record native DESI mass spectra for all three with masses of 66, 148 and 800 kDa, respectively and with established subunit stoichiometries ( Figure 1d,e,f). These results effectively transform DESI from as mall molecule approach to am ethod capable of detecting intact protein assemblies from surfaces.I nt his regard an obvious next target is membrane proteins since their natural environment is in lipid bilayers.
Forthis study we selected the outer-membrane protein F (OmpF), atrimer of transmembrane beta-barrels.W edeposited 0.4 nmol of OmpF in ammonium acetate (200 mm) containing octyl glucoside (OG) micelles and directed the desorption plume at the deposited protein. Initially ar elatively low intensity signal was observed (Figure 2a)followed by rapid deterioration and loss of signal-indicating adilution effect at the surface leading to disruption of the micelle. [18] Adding OG to the desorption solution (at twice the critical micelle concentration (cmc) (1 %w /v OG) we observed recovery of the OmpF trimer signal (Figure 2b)w hich remained stable for 30 mins ( Figure S2). [18] Substituting ad ifferent detergent (Lauryldimethylamine N-oxide (LDAO)0 .05 %w /v) into the desorption plume induces as hift to higher charge states (Figure 2c), observed previously with LDAO [19] implying that detergent exchange has occurred on the stage.This highlights an important capability of the native DESI approach for rapid detergent screening.
Screening for optimal detergents,a sp art of membrane protein purification protocols,i st ime consuming and uses valuable protein resources. [20] We investigated further the possibility of detergent screening on the DESI stage using the sugar transporter semiSWEET from Vibrio splendidus [21] since it is extremely sensitive to its detergent environment. Previous MS experiments established that this transporter exists in an entirely monomeric form in DDM and exhibits am onomer-dimer equilibrium in the detergent (C8E4). [22] Depositing semiSWEET on the DESI stage in DDM (Figure 2d)w et hen added C8E4 (0.5 %w /v) directly to the desorption buffer.After 1.8 min of desorption with the C8E4containing buffer the total ion chromatogram for the (7 +)ion (indicative of the dimer) was observed consistent with detergent exchange on the DESI stage from the initial DDM conditions to the C8E4 micelles (Figure 2e). Detergent screening on the DESI stage,with minimal membrane protein consumption, highlights ap owerful feature of this approach.
Tu ring to ligand screening an important criterion to establish is the extent to which protein complexes dissociate into their components during native DESI as opposed to conventional nanoflow ES.S electing an outer membrane protein receptor FpvA from Pseudomonas aeruginosa, which translocates ferric-pyoverdine (Pvd) across outer mem- Figure 1. Schematic of the native DESI setup showing deposition of protein on the stage, followed by desorption and analysis in the mass spectrometer with representative spectra for aseries of soluble protein their substrates and complexes. a) First, protein is deposited on the native DESI stage (red) from aqueous buffer and second, the ES plume is charged with avoltage of 2.5-3.5 Vand directed at the stage. Transfer is effected by positioningthe stage close to the orifice of the mass spectrometer.b)Apo lysozymei sdeposited on the stage (25 mL, 10 mm)in aqueous ammonium acetate (200 mm,p H6.8) and the same buffer is used to desorb the protein. c) NAG-5 is added to the ES plume directed at the lysozyme deposit, additionalp eaks reveal binding of the substrate NAG-5 prior to its cleavage. Native DESI mass spectra of d) monomeric bovine serum albumin, e) tetrameric alcohol dehydrogenase and f) the GroEL 14-mer. branes, [23] we formed the complex in solution and compared the percentage of complex FpvA:PvD using the different ionization methods.W ef ound 59 %a nd 61 %i nt he native DESI and nanoES approaches,r espectively (Figure 3a and b). We conclude that our native DESI approach, which involves both deposition and desorption within protective micelles,r eproduces the nano-ES result in which complexes are electrosprayed directly from solution.
Exploring further the quantitative aspects of this native DESI platform we selected OBS1 a17-residue peptide known to bind within the pores of OmpF with binding constants determined previously by both ITC and nanoES MS. [24,25] We deposited OmpF on the stage in OG detergent and incubated OmpF with increasing concentrations of OBS1 (0-75 mm)and deposited these protein-peptide complexes onto the native DESI stage.T he relative intensities of bound to unbound protein were extracted and plotted as af unction of OBS1 concentration ( Figure S3). The K d determined (0.7 AE 0.34 mm) for this membrane protein complex is in agreement with values reported (1.0 AE 0.1 mm). [24] That the solution state equilibria is maintained is surprising given that DESI involves ejection of the membrane protein complex, deposited on the surface,b yadesorption spray.H owever detailed kinetics of OmpF-OBS1 peptide complexes are not known so it is unclear how this reflects the kinetics of DESI.
Beyond peptide binding established in solution we wanted to perform binding experiments on membrane proteins deposited on the stage.D elipidated apo OmpF (20 mm)w as deposted and OBS1 added to the desorption buffer. Up to three peptides bound per trimer were detected with the predominant peak corresponding to two (Figure S3 a). This experiment confirms that the peptide can access binding sites within the short time frame during desorption, even in the presence of detergent. Similarly we added phosphatidylglycerol lipid (POPG) to the desorption buffer, at ac oncentra-tion of 5 mm,and observed up to three lipids bound to OmpF (Figure S3 b). OmpF is also reported to bind to ar ange of antibiotics within the extracellular and periplasmic pore vestibule. [26] Addition of kanamycin (50 mm)tothe desorption buffer reveals binding of one molecule of the antibiotic per OmpF trimer (Figure S3 c). Since we anticipate that the peptide binds within the pore,w hile the lipids bind to the outer surface and kanamycin to the top of the pore,wehave highlighted our capability to bind directly to the membrane protein via three different mechanisms.Ineach of these three scenarios we assume that the small molecule has not reached solution phase equilibrium, since desorption is rapid, but rather has penetrated to some extent the protective micelle that surrounds the membrane protein while deposited on the surface.

Communications
Building on this ability to screen for binding to am embrane protein target deposited on asurface we reasoned that it would be possible to add multiple ligands simultaneously. To explore this possibility we selected aC lass AG -protein couple receptor (GPCRs) depositing onto the stage 0.4 nmoles of P2Y 1 ,r esponsible for platelet aggregation and ak ey target for anti-thrombotic therapy. [27] After recording anative DESI spectrum in its apo form we added acocktail of antagonists/agonists designed to target related GPCRs (Figure 3d,e and Table S1). Then ative DESI spectrum reveals adiscrete mass increase (560.03 Da) in exact agreement with the mass of MRS2500 (1'R,2'S,4'S,5'S)-4-(2-Iodo-6-methyl amino-purine-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicycle[3.1.0]-hexane bound to P2Y 1 .N oo ther adducts were observed and control experiments,w here the specific inhibitor was excluded from the drug cocktail, revealed no ligand binding to P2Y 1 ( Figure S6). These results reveal that this native DESI platform is capable of detecting selective binding of as pecific antagonist to aG PCR from amulticomponent mixture.
In summary we have developed and applied an ative DESI platform and shown that it is capable of preserving the native structure of both soluble and membrane proteins and their complexes.C omparing our approach with ambient ionization methods described previously we note the addition of chemicals in the spray solution in reactive DESI applications for small molecule analyses. [17,28] Protein ligand binding experiments have also been achieved with reactions taking place by mixing in solution in al iquid sample DESI approach [6] rather than by interaction following protein deposition on the planar target as shown here.M oreover kinetic approaches have been developed using mixing experiments to monitor the small molecules released during enzymatic cleavage by means of liquid DESI and have increased the range of buffers that can be used. [29,30] Our native DESI approach is largely restricted to volatile buffers and detergents that have been optimised for native MS.A further limitation is imposed by the fact that ligands are observed directly bound to proteins desorbed from ap lanar surface,h igh-resolution MS is therefore critical.
Them ost exciting aspect of this native DESI approach however, is the potential of our method to study intact membrane proteins and their complexes.T he ability to place membrane protein targets on planar surfaces in different lipidic environments,w ithout tethering the proteins,a nd to carry out selective binding from ac ocktail of drugs offers possibilities for high throughput screening.Many downstream applications become accessible including the ability to carry out multiple experiments on the same target;f or example detergent optimisation, the screening of multiple lipids and ligands that bind to ad rug target or the trapping of fast turnover products in enzyme catalysed reactions.A nalogous to the powerful native MS methods,n ow widely accepted as ak ey component in structural biology,n ative DESI enables further possibilities for development of spatial, temporal and even directional analyses within artificial bilayers or membrane mimetics.