Covalent Cross-Linking as an Enabler for Structural Mass

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Covalent Cross-Linking as an Enabler for Structural Mass Spectrometry Emeline Hanozin, Elodie Grifnée, Hugo Gattuso, Andre Matagne, Denis Morsa, and Edwin De Pauw Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02491 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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Analytical Chemistry

Covalent Cross-Linking as an Enabler for Structural Mass Spectrometry Emeline Hanozin,1 Elodie Grifnée,1 Hugo Gattuso,2 André Matagne,3 Denis Morsa,1 Edwin De Pauw1* 1

Mass Spectrometry Laboratory, Molsys Research Unit, University of Liège, B-4000 Liège,

Belgium 2

Theoretical Physical Chemistry, Molsys Research Unit, University of Liège, B-4000 Liège,

Belgium 3

Laboratory of Enzymology and Protein Folding, Center for Protein Engineering, University

of Liège, B-4000 Liège, Belgium

AUTHOR INFORMATION Corresponding Author *Prof. Edwin De Pauw. Chemistry Building (B6c), Quartier Agora, Allée du Six Août, B-4000 Liège, Belgium. Email: [email protected] Notes The authors declare no competing financial interest.

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ABSTRACT Studies referring to the structural elucidation of intact biomolecular systems using mass spectrometry techniques have been gradually flourishing in the post-2000s literature topics. As part of native mass spectrometry, this domain capitalizes on the kinetic trapping of physiological folds in view of probing solution-like conformational properties of isolated molecules or complexes after their electrospray transfer to the gas phase. Despite its documented efficiency for a wide array of analytes, this approach is expected to be pushed to its limits when considering highly dynamic systems or when dealing with non-ideal operating conditions. To circumvent these limitations, we challenge the adequacy of an original strategy based on cross-linkers to improve the gas-phase stability of isolated proteins and ensure the preservation of folded conformations when measuring with strong transmission voltages, by spraying from denaturing solvents or trapping for extended periods of time. Tested on cytochrome c, myoglobin and β-lactoglobulin cross-linked using BS3, we validated the process as a structurally non-intrusive in solution using far-ultraviolet circular dichroism and unraveled the preservation of folded conformations showing better resilience to denaturation on crosslinked species using ion mobility. The resulting collision cross sections were found in agreement with the native fold, and a preservation of the proteins’ secondary and tertiary structures was evidenced using molecular dynamics simulations. Our results provide new insights concerning the fate of electro-sprayed cross-linked conformers in the gas phase, while constituting promising evidence for the validation of this technique as part of tomorrow’s structural mass spectrometry workflows.


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INTRODUCTION A key in the understanding of biological processes and mechanisms occurring in life sciences lies in determination of the functions of the involved biomolecules, among which proteins constitute essential actors. From the assumption that these functions are closely related to the tridimensional conformations of such molecules,1 the development of adequate tools to unravel their structural properties is of paramount priority. Although historically covered by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, the advent of “soft” electrospray ionization (ESI) sources in the late 80s set a milestone in the application of mass spectrometry (MS) for structural biology. Circumventing purity and size-related issues, and capitalizing on their rapidity, sensitivity and selectivity, MS methods dedicated to protein analysis have been developed toward two complementary axes to resolve either sequencerelated properties on individual proteins, i.e. proteomics MS,2 or topologically-related properties on both individual proteins and complexes, i.e. native MS.3,4 In its strictest definition, native MS refers to the electrospray ionization of biomolecules from physiological solvent conditions.5 These dispositions set the preliminary requirements leading to the preservation of noncovalent interaction networks during the gas-phase transfer,6,7 so that intact proteins and biomolecular complexes may be probed in their biological state.8 Through preservations, essential information related to the stoichiometries, topologies and spatial organizations of fragile assemblies such as viruses,9 membrane proteins, antibodies, and even ribosomal subunits10 sizing up to a few MDa11 are reachable using MS. Additionally, the resulting molecular ions may be further interrogated by MS-hyphenated techniques such as action spectroscopy,12 ion mobility (IM)13,14 or collision-induced unfolding (CIU)15,16 preceding dissociation to provide supplementary structural data. While the stabilizing intramolecular interactions network offers some guarantees to retain some of the physiological fold features in the gas phase,6,17,18 the preservation of 3 ACS Paragon Plus Environment


solution-like conformations is still a result of kinetic trapping.19,20 Their refolding into alternative thermodynamically-favored gas-phase conformations21 are therefore conditioned by their conformational landscapes, i.e. “how high the rearrangement barriers are”, their internal energy content, i.e. “how much energy is available to overcome these barriers”,22,23 and the experiment timescale, i.e. “how much time is left for the energy to build up”.24,25 Consequently, probing physiologically-relevant structural properties by MS when dealing with highly dynamic systems associated with shallow folding funnels,26 when using sensitivityimproving higher transmission voltages or when interrogating over long > 100 ms timescales may constitute challenging tasks possibly leading to biased data. The use of covalent cross-linkers (CL) has been recently evoked by Samulak et al. as a strategy to enhance the gas-phase stabilities of large multi-protein complexes and ensure that their constitutive subunits remain folded when probed using collision-induced dissociation (CID) experiments in MS.27 In the present study, we capitalize on this approach and further investigate the adequacy of the cross-linking strategy as a methodology to overcome possible limitations inherent to native structural MS workflows when dealing with uncommon analytes or non-ideal operating conditions. Three aspects are to challenge: (i) the structural effect induced by cross-linking on the biomolecular conformation in solution prior to MS analysis, (ii) the extent of correlation found between the physiological fold and the cross-linked gasphase fold after ESI transfer and (iii) the trapping efficacy offered by cross-linkers toward the preservation of folded structures under denaturing solvents, harsh MS experimental conditions and long probing timescales. We evaluate these using small protein models, i.e. cytochrome c (Mw = 12.4 kDa), myoglobin (Mw = 16.7 kDa) and β-lactoglobulin (Mw = 18,4 kDa), whose gas-phase conformations are documented as plural and highly sensitive to instrumental settings, trapping times, solvent conditions and charge states.22,24,28 The reaction products


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achieved using bis(sulfosuccinimidyl)suberate (BS3) as a linker reagent29 were structurally investigated using circular dichroism (CD) in solution and using IM-MS and CIU in gas phase. While our results corroborate existing studies that suggest minimal structural impact after cross-linking in solution,30 significant discrepancies in the conformational landscapes between non-cross-linked and cross-linked species could be monitored in the gas phase under certain conditions. These results materialized through a delayed apparition of elongated unfolded structures toward higher transmission voltages and longer trapping times after implanting cross-linkers. The ability to preserve folded conformations is directly dependent on the protein charge state, solvent conditions, instrumental settings and content in cross-linkers. These results are interpreted in accordance with physiologically-compliant benchmarks issued from nuclear magnetic resonance (NMR) and discussed based on molecular dynamic (MD) simulations.

EXPERIMENTAL SECTION Chemicals. Horse heart cytochrome c, bovin milk β-lactoglobulin, horse heart myoglobin and ammonium bicarbonate (NH4HCO3) were purchased from Sigma Aldrich (Belgium). UPLC/MS grade absolute methanol (MeOH) was purchased from Biosolve (the Netherlands). Dulbecco's phosphate-buffered saline (DPBS) was purchased from BioWhittaker Lonza (Belgium). Bis(sulfosuccinimidyl) suberate (BS3) conditioned in 2 mg microtubes was obtained from ThermoFisher Scientific (Waltham, MA, USA). All reagents were used without further purification. Cross-linking Reactions. Cytochrome c, β-lactoglobulin and myoglobin were dissolved to a final concentration of 20 µM in Dulbecco's Phosphate-Buffered Saline (DPBS) solution (pH 7.4). BS3 cross-linking reagent was first dissolved in a small amount of milli-Q water (2 mg/50 µL) and then diluted in DPBS to a final concentration of 7 mM. All samples were cross-linked



at 25 °C under stirring at 600 rpm (Thermomixer comfort, Eppendorf, Hamburg, Germany). Cytochrome c samples were cross-linked at 40x, 100x and 200x molar excess of BS3 for 2 min, 30 min and 2 h respectively. β-lactoglobulin and myoglobin were both cross-linked at a 40x molar excess of BS3 for 3 min, 5 min, 30 min, 2 h and 5 h. The cross-linking reactions were quenched by adding NH4HCO3 1 M to a final concentration of 20 mM. The cross-linked samples were then washed twice with NH4HCO3 500 mM and twice with NH4HCO3 25 mM on Amicon Ultra (3K, 0.5 mL) centrifugal filters (Merck Millipore, Ireland) centrifuged at 1200 rpm (Eppendorf Centrifuge 5415 R) according to the manufacturer’s protocol. The resulting cross-linked samples were diluted to a concentration of ~10 µM using 10 mM ammonium acetate (NH4Ac) aqueous buffer (buffer) and methanol (MeOH) mixed either in 100:0 (physiological conditions) or 25:75 (denaturing conditions) ratios. Circular Dichroism Measurements. Far-UV CD spectra (185-260 nm) were recorded with a Jasco J-810 spectropolarimeter at 20 °C using a 1 mm pathlength quartz Suprasil cell (Hellma). Proteins were dissolved in either 100:0 buffer:MeOH or in 25:75 buffer:MeOH to a reach a concentration of ca. 0.1 mg/mL. Four scans (20 nm/min, 1 nm bandwidth, 0.1 nm data pitch and 2 s digital integration time DIT) were averaged, base lines were subtracted, and no smoothing was applied. Data are presented as the molar residue ellipticity [θ] MRE calculated based on the molar concentration of proteins and number of residues. Secondary structure analyses using the CDSSTR,31,32 CONTINLL33,34 and SELCON335,36 algorithms were performed on the CD data with the CDPro software package,32 using reference datasets SP27, SP39, SP43. Ion Mobility Mass Spectrometry Measurements. Ion mobility consists in separating ions based on their mobility coefficient K that is dependent on their charge-to-collision cross section ratio z/Ω.37 CIU-related measurements were performed on a SYNAPT G2-Si HDMS spectrometer (Waters, Manchester, UK)38,39 fitted with an ESI source and using N2 in the 6 ACS Paragon Plus Environment

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mobility cell. Instrumental settings were set as follows to minimize activation: capillary voltage = 1.6 kV, sampling cone = 5 V, source offset = 1 V, source temperature = 80 °C, desolvation temperature = 100 °C, trap CE = 2 V, transfer CE = 2 V. CIU experiments were performed by varying the trap bias voltage between 35 V and 100 V. Trapping-related measurements were performed on a timsTOF spectrometer (Bruker Daltonics, Bremen, Germany) fitted with an ESI source and using N2 in the mobility cell.40,41 Instrumental settings were set as follows to minimize activation : capillary voltage = 4.5 kV, end plate offset = 2.5 kV, dry temperature = 220 °C, Δ1 = -20 V, Δ2 = -270 V, Δ3 = 18 V, Δ4 = 250 V, Δ5 = 0 V and Δ6 = 70 V. Accumulation times, prior to the mobility separation, were varied between 20 ms and 300 ms. Complete setups are provided in the Supporting Information, Table S1 (Synapt G2-Si) and Table S2 (timsTOF). Collision Cross Section Calibration. For the Synapt G2-Si HDMS (Waters), the calibration of travelling wave drift times to He-related collision cross sections TWCCSN2He was established following the methodology described by Ruotolo et al.38 The list of used calibrants and the resulting calibration curve are reported in the Supporting Information, Table S3 and Figure S1 respectively. For the timsTOF (Bruker), the calibration of mobility coefficient 1/K0 to N2related collision cross sections

TIMS

CCSN2 was performed using the low-concentration tuning

mix purchased from Agilent Technologies (Santa Clara, CA) with the following standards : m/z = 622, 1/K0 = 0.985 V.s/cm2; m/z = 922, 1/K0 = 1.190 V.s/cm2; m/z = 1222, 1/K0 = 1.382 V.s/cm2; m/z = 1522, 1/K0 = 1.556 V.s/cm2; m/z = 1822, 1/K0 = 1.729 V.s/cm2.42 Standard deviations are determined from the 95% confidence interval. The formalism for the notation of the collision cross section is described elsewhere.43 Collision Cross Section Theoretical Calculation. Theoretical collision cross sections of NMR and X-Ray resolved structures, later referred to as CCSNMR and CCSX-Ray, were computed using an improved exact hard sphere scattering (EHSS) model44 and corrected collision radii,45 7 ACS Paragon Plus Environment


as implemented in the EHSSrot software.46 For cytochrome c, CCSNMR = 1317 ± 11 Å2 from the averaged values of 18 independent CCSNMR corresponding to each available structure (PDB: 1AKK, 1OCD, 2N3B). For β-lactoglobulin, CCSNMR = 1821 ± 25 Å2 (PDB: 1CJ5, 1DV9) and for myoglobin, CCSX-Ray = 1708 ± 24 Å2 (PDB: 1WLA). Collision induced unfolding heatmaps. CIU heatmaps were generated from experimental data using the CIUSuite 2 software available on the Ruotolo Research Group website.16 Molecular dynamics simulations. The Amber47 ff14SB force field48 as available in the AMBER program suite was used for simulations. Specific force field parametrizations related to the heme group (total charge = -1, doublet spin multiplicity) and the BS3 linker were implemented by optimizing their corresponding geometries in Gaussian09 D01 software49 at the DFT(B3LYP)/LANL2DZ (heme) and DFT(B3LYP)/6-31+G(d) (BS3) levels of theory. After optimization, partial atomic charges were derived for both entities using the standard RESP procedure.50 (Supporting Information, Figure S2). The NMR-derived structure of cytochrome c (PDB: 1AKK), with C-ter N-Me amide capping groups and neutral forms of Glu and Asp residues, was used as an initial geometry. The MD protocol starts with a geometry relaxation stage including 4000 steps with the steepest descent algorithm followed by 4000 steps with the conjugate gradient algorithm. Then, a thermalization stage is performed followed by a 500 ns long production run at 300 K. The time step of integration is 2 fs. Two distinct thermalization stages were considered: (a) a “mild” thermalization, during which the temperature is quickly increased from 0 K to 300 K for 200 ps and (b) a “harsh” thermalization consisting of a 400 ps long stepwise heating up to 600 K followed by a 20 ps cooling down to 300 K. All simulations were performed in the gas phase so that neither periodic boundary conditions nor Ewald summation for electrostatic interactions are required.


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RESULTS AND DISCUSSION Characterizing the products of a cross-linking reaction by mass spectrometry. The covalent linkage of a target protein engenders diverse products consisting of differentially mono-linked (ML) species, where only one side of the reactant covalently binds to the protein, and cross-linked (CL) species, where both sites of the reactant get attached and establish an intramolecular link between two targeted amino acid residues of the protein. These species are efficiently separated using MS as illustrated in Figure 1a, which provides the MS spectrum monitored after a 2-min incubation of cytochrome c with BS3 in 40x molar excess. The spectrum is firstly shaped by bunches of peaks respectively associated with different contents in attached linkers, e.g. here from 1 to 5 linkers. Within a given bunch, distinct isotopic patterns are present and correspond to variable stoichiometries of a same linker content spread through ML and CL components, as illustrated in Figure 1d for four linkers. The patterns are split by an 18 Da mass increment due to the inactivated carboxyl group at one extremity of ML. The extent of covalent linkage may be efficiently modulated through the linker molar excess and the reaction duration. This feature is illustrated in Figure 1a, 1b, and 1c which show a progressive shift toward highly linked products, e.g. containing up to 9 concomitant CL for cytochrome c, when the BS3 molar excess and the reaction times are increased from 40x to 200x, and from 2 min to 2 h respectively. Similar behaviors were monitored for β-lactoglobulin, which displays a maximum of 4 concomitant CL, and for myoglobin which yet tends to promote the formation of ML products (Supporting Information, Figure S3). All observations are in good agreement with results reported in the literature that were assigned using a bottom-up identification workflow.51–54



Figure 1. Mass spectra monitored on cytochrome c (z = +7) sprayed from physiological solvent (100:0 buffer:MeOH) after cross-linking with BS3 for (a) 40x linker molar excess and 2 min, (b) 100x linker molar excess and 30 min and (c) 200x linker molar excess and 2 h. The spectra display packs of peaks associated with a defined number of attached linkers. The distribution is shifted toward higher linker contents as the linker concentration and reaction time increase. (d) Within a given pack, distinct isotopic patterns coincide with different stoichiometries of ML and CL content: 4 CL (red), 3 CL + 1 ML (blue), 2 CL + 2 ML (green) and 1 CL + 3 ML (orange). Instrument was operated in Q-TOF mode with IM separation off. Assessing the impact of cross-linking on the protein conformation in solution. Circular dichroism (CD) measurements were performed to validate the conformational integrity of proteins, i.e. the absence of significant structural rearrangement, following their covalent cross-linking in solution. To this end, the secondary structure of both the non-cross-


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linked sample and the highly cross-linked (200x BS3 molar excess and 2 h reaction time) sample of cytochrome c were probed in physiological (100:0 buffer:MeOH) and denaturing (25:75 buffer:MeOH) solvents. Figure 2a shows the evolution of the molar residue ellipticity [θ] MRE of the two predefined samples between 185 nm and 260 nm in physiological solvent. The two samples exhibit identical CD profiles characterized by two minima at 208 nm and 222 nm, evidencing the presence of α-helical folds inside the protein structure. Contents in secondary structures of the non-cross-linked and highly cross-linked samples were respectively estimated as 41.1 ± 0.6 % and 43.5 ± 0.3 % α-helical in physiological conditions, which is in good agreement with data reported in the Protein Data Bank (PDB) (~39 % α-helical for 1AKK). Switching to denaturing solvent, both CD profiles account for higher α-helical contents equal to 47.5 ± 1.2 % and 45.2 ± 0.9 % respectively. This increase can be associated with the presence of methanol and correlates with the strengthening of polar interactions, promoting the formation of secondary structures, and with the weakening of hydrophobic interactions, inducing an expansion of the hydrophobic core.55 While no significant reshape in the CD profile is monitored for the highly cross-linked cytochrome c sample between physiological and denaturing solvents (Figure 2b), significant variations underlining substantial structural reorganizations are observed between 200 nm and 220 nm for the non-cross-linked sample (Figure 2c). Overall, CD results emphasize two aspects related to the covalent linkage in solution: (a) the process is structurally non-intrusive as the content in secondary structures is barely affected by the presence of linkers, and (b) the resulting cross-linked structure displays sustained conformational stability in denaturing solvent compared to the non-cross-linked one.



Figure 2. Molar residue ellipticity [θ] MRE as a function of the wavelength for the non-crosslinked and cross-linked cytochrome c samples as recorded from far-UV CD measurements. (a) CD profiles of both samples monitored in physiological solvent (100:0 buffer:MeOH). (b-c) Overlays of CD profiles monitored in physiological (100:0 buffer:MeOH) and denaturing (25:75 buffer:MeOH) solvents for the (b) cross-linked and (c) non-cross-linked cytochrome c samples.

Charge state distributions to gauge the ions compactness during the gas-phase transfer. The charge-state distribution (CSD) layouts constitutive of ESI-MS spectra are directly dependent on the compactness of the corresponding molecular ions during their transfer to the gas phase.56,57 Typically, the more unfolded a conformation, the more it is exposed to solvent protonation and the higher are the resulting charge states. Figure 3 reports the CSDs monitored for the non-cross-linked, lightly cross-linked (3 to 5 CL) and heavily cross-linked (8 to 9 CL) cytochrome c samples sprayed from physiological (100:0 buffer:MeOH) and denaturing (25:75 buffer:MeOH) solvents. In physiological solvent, 12 ACS Paragon Plus Environment

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the CSDs are all centered on z = +7 and z = +8 regardless of the content in linkers, which is within adequacy with existing data obtained using “soft” instrumental settings.28 In denaturing solvent, the apex of the CSD monitored for the non-cross-linked sample is shifted toward higher charge states, i.e. z = +13 and z = +14, therefore revealing substantial structural unfolding occurring during the electrospray process. This phenomenon is prevented when the protein is covalently cross-linked prior to the gas-phase transfer, from partial extents at low CL content to full extents at high CL content for which the z = +7 and z = +8 charge states still largely dominate the CSD. Similar behaviors were observed for β-lactoglobulin and myoglobin (Supporting Information, Figure S4). Intriguingly, our results also show that similar charge states are achievable for both cross-linked and non-cross-linked samples, despite the reduction in the number of vacant primary amines consecutive to the cross-linking reaction. These observations corroborate models arguing that the extent of multiple charging of protein ions achieved by ESI is mainly conditioned by the solvent-exposed surface area rather than by intrinsic factors such as the number of basic residues.58



Figure 3. CSDs monitored in Q-TOF mode (IM off) for (a) the non-cross-linked sample, (b) the lightly cross-linked sample (from 3 to 5 CL) and (c) the highly cross-linked sample (from 8 to 9 CL) of cytochrome c sprayed from physiological (100:0 buffer:MeOH) and denaturing (25:75 buffer:MeOH) solvents.

Gas-phase conformations of cross-linked proteins: influence of the energetics. With the assessed contribution of cross-linkers that aid in maintaining folded conformations during the electrospray process, we further used ion mobility to probe their influence on the conformational fate of model molecular ions after they settled for a few ms (< 20 ms) in the gas phase. These measurements were performed in a traveling-wave mobility cell on the SYNAPT G2-Si (Waters) for different trap bias voltages ranging from 35 V, i.e. “nativecompliant”, to 100 V, i.e. “CIU-promoting”.59 Figure 4a and 4b illustrate the CIU heatmaps generated for, respectively, the z = +7 and z = +8 ions of cytochrome c bearing 0 CL, 2 CL and 9 CL and sprayed from physiological solvent (100:0 buffer:MeOH). Our results show that a gradual increase in the trap bias voltage on the non-cross-linked species induces sequential shifts in the maximum intensity spots. For z = +7, this phenomenon is characterized by a first ~1400 Å2 to ~1700 Å2 transition at 40V, and a second ~1700 Å2 to ~1850 Å2 transition at 50 V, together with the concomitant apparition of a less intense conformer at 2100 Å2 starting from 70 V. For z = +8, a single ~1800 Å2 to ~2150 Å2 transition occurs at 50 V. Repeating a similar process after covalent linkage results in a reshape of the heatmaps manifested by shifts in the conformational transitions toward stronger voltages for low linker contents (2 CL) and by the retention of the most compact conformers along the entire probed voltage range for high linker contents (9 CL). Detailed TWCCSN2He distributions monitored for both z = +7 and z = +8 molecular ions sprayed from physiological solvent (100:0 buffer:MeOH) and bearing from 0 to 9 CL were extracted for the lowest trap bias voltage of 35 V (Figure 4c and 4f), for an intermediary trap 14 ACS Paragon Plus Environment

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bias voltage of 60 V (Figure 4d and 4g) and for the highest trap bias voltage of 100 V (Figure 4e and 4h). The distributions achieved after spraying from denaturing solvent (25:75 buffer:MeOH) are also provided for the intermediary trap bias voltage. The red line mark is associated with the theoretical CCS of the NMR-derived physiological structure in solution. To facilitate the discussion, conformational zones were delimited and labeled from A to E following the nomenclature proposed by Badman et al.24,25: A from 1317 Å2 to 1530 Å2, B-C from 1530 Å2 to 1750 Å2, D from 1750 Å2 to 2000 Å2 and E from 2000 to 2220 Å2. First discussing z = +7 sprayed from physiological solvent,

TW

CCSN2He = 1380 ± 20

Å2 is measured for the non-cross-linked species at the lowest trap bias, which is within adequacy of recent native MS data reported in the literature60 and only 5% higher than CCSNMR = 1317 ± 11 Å2. After cross-linking, a slight gradual compaction is monitored leading to a TW

CCSN2He = 1350 ± 16 Å2 for 9 CL. Switching to intermediary trap bias, the non-cross-linked

species display a major conformer in zone D, i.e. TWCCSN2He = 1799 ± 18 Å2 and two minor conformers in zone B-C, i.e. TWCCSN2He = 1666 ± 17 Å2, and in zone E, i.e. TWCCSN2He = 2139 ± 19 Å2. After cross-linking, populations are gradually reshaped through the extinction of conformer E after the addition of 1 CL, the equaling in the population of conformer B-C and conformer D after addition of 2 CL and the apparition of a compact conformer A after the addition of 2 CL. The latter becomes the major conformer starting from 3 CL and largely dominates the distribution after the addition of 9 CL with a resulting TWCCSN2He = 1479 ± 16 Å2. Finally, focusing on the highest trap bias, the non-cross-linked species display two major conformers already highlighted at 60 V: conformer D with conformer E with

TW

CCSN2He = 1799 ± 18 Å2 and

TW

CCSN2He = 2139 ± 19 Å2. Cross-linking results in similar outcomes

materialized by the extinction of conformer E and the apparition of a compact conformer first located in zone B-C from 1 CL to 5 CL and eventually reaching a TWCCSN2He = 1480 ± 20 Å2 in zone A for 9 CL. Similar reshapes in the populations, resulting in compact conformers are



monitored when spraying from denaturing solvent, although the initial population of the noncross-linked species are further moved to conformer E. Switching to z = +8, higher Coulomb repulsions61 favor conformer D with TWCCSN2He = 1820 ± 19 Å2 for the non-cross-linked species at the lowest trap bias. Other minor conformers are present in zone B-C and in zone E. After the addition of 1 CL to 3 CL, the population of conformer D decreases in favor of a more compact conformer localized in zone B-C at TW

CCSN2He = 1555 ± 18 Å2. The most compact conformer belonging to zone A is formed

starting from 5 CL and reaches

TW

CCSN2He = 1484 ± 17 Å2 after the addition of 9 CL. Non-

cross-linked populations are similar for the intermediary and highest trap bias voltages with a major conformer localized in zone E at localized in zone D at

TW

CCSN2He = 2125 ± 20 Å2 and a minor conformer

TW

CCSN2He = 1925 ± 20 Å2. After cross-linking, the population shifts

from conformer E toward an emerging compact conformer localized in zone B-C to reach TW

CCSN2He = 1589 ± 18 Å2 after the addition of 9 CL. The results show that, for z = +7, conformers with similar CCSs were monitored for all

species bearing from 0 CL to 9 CL when using “native-compliant” low trap bias voltages. This observation suggests that, with minimal energetic stress, cross-linked species adopt similar conformations as non-cross-linked species, and are therefore relevant of the native fold. When using harsher energetic conditions, when spraying from denaturing solvent or when considering higher charge states favoring Coulomb repulsions, the non-cross-linked species unfold, and the population is shifted toward elongated conformers. This phenomenon is gradually prevented as the content in cross-linkers increases to achieve almost-single compact conformers lying in zone A when the maximum number of 9 CL is embedded. Similar reshapes in the population of the z = +9 molecular ions of both β-lactoglobulin and myoglobin bearing from 0 CL to 4 CL were monitored considering an intermediary trap bias voltage of 60 V (Supporting Information, Figure S5).


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Figure 4. CIU heatmaps showing the conformational populations of the (a) z = +7 and (b) z = +8 molecular ions of cytochrome c sprayed from physiological solvent and bearing 0 CL, 2 CL



and 9 CL for trap bias voltages ranging from 35 V to 100 V. TWCCSN2He distributions recorded for z = +7 at trap bias of (c) 35V, (d) 60 V and (e) 100 V and for z = +8 at trap bias of (f) 35V, (g) 60 V and (h) 100 V for species bearing 0 CL, 1 CL, 2 CL, 3 CL, 5 CL and 9 CL sprayed from physiological solvent (100:0 buffer:MeOH, solid line) and denaturing solvent (25:75 buffer:MeOH, dashed line). Conformational zones are delimited as follows: A from 1317 Å2 to 1530 Å2, B-C from 1530 Å2 to 1750 Å2, D from 1750 Å2 to 2000 Å2 and E from 2000 to 2220 Å2.

Gas-phase conformations of cross-linked proteins: influence of the trapping time. Long storage times were proved to increase the likelihood of conformational transitions.62 In this context, previous studies highlighted the formation of elongated conformations for ubiquitin when stored over 1 s62 as well as for cytochrome c starting from ~50 ms trapping times.24,25 Here, we analyzed the result of cross-linking on the conformational landscape of the z = +8 molecular ion of cytochrome c sprayed from denaturing solvent (25:75 buffer:MeOH) when stored for extended periods. These measurements were performed on the timsTOF spectrometer (Bruker) for different trapping times ranging from 50 ms to 300 ms and occurring before the mobility separation. As the TIMS calibration was performed with CCS measured in N2, the previously used conformational zones were updated considering the mathematical description of the CCS within the hard-sphere limit63: A from 1371 Å2 to 1589 Å2, B-C from 1589 Å2 to 1813 Å2, D from 1813 Å2 to 2068 Å2, E from 2068 to 2291 Å2 and F beyond 2291 Å2. Figure 5 shows that the

TIMS

CCSN2 distribution of the non-cross-linked species is

centered on two major conformers localized in zone D, i.e. TIMSCCSN2 = 1896 ± 4 Å2, and in zone E, i.e. TIMSCCSN2 = ~2145 ± 4 Å2 after a 50 ms trapping. These populations get reshaped in favor of conformer E after a 100 ms trapping, and toward the production of highly unfolded conformers belonging to zone F after a 300 ms trapping. By incorporating gradually increasing 18 ACS Paragon Plus Environment

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content in cross-linkers, the distributions get progressively shaped in favour of more compact emerging conformers to eventually reach the zone B-C with a TIMSCCSN2 = 1770 Å2 for 9 CL. This one still largely dominates after 300 ms although a less abundant fraction of conformer E is concomitantly formed. This result accounts for the ability of CL to consistently maintain compact folded conformations for extended trapping times of up to several hundred ms in the gas phase.

Figure 5. TIMSCCSN2 distributions recorded after (a) 50 ms, (b) 100 ms and (c) 300 ms trapping times for the z = +8 molecular ion of cytochrome c sprayed from denaturing solvent (25:75 buffer:MeOH) and bearing 0 CL, 1 CL, 2 CL, 3 CL, 5 CL and 9 CL. Conformational zones are



delimited as follows: A from 1371 Å2 to 1589 Å2, B-C from 1589 Å2 to 1813 Å2, D from 1813 Å2 to 2068 Å2, E from 2068 to 2291 Å2 and F beyond 2291 Å2. Molecular dynamics simulations as a conformational probe at the atomic scale. Molecular dynamics (MD) simulations were performed to get a description of the observed phenomena at the atomistic scale and to provide insights regarding the extent of correlation evidenced between the native fold and the cross-linked fold in the gas phase. To this end, two starting geometries of cytochrome c bearing 0 CL and 8 CL were designed considering z = +8. The position of the linkers were established as follows based on literature data51,52,54,64,65 and avoiding major steric constrains: (1) Lys-7 – Lys-100, (2) Lys-13 – Lys-8, (3) Lys-25 – Lys-27, (4) Lys-39 – Lys-53, (5) Lys-72 – Lys-79, (6) Lys-86 – Lys-73, (7) Lys87 – Lys-88 and (8) Lys-99 – Lys-60 (Supporting information, Figure S6 and Table S4). In addition to the formal positive charge carried by the heme group, 7 sites of the protein were protonated considering their respective basicity in solution and aiming at minimizing Coulomb repulsions (Supporting information, Figure S7 and Table S5). Both thermalization stages at 300 K and at 600 K were considered prior to the MD production runs to respectively mimic “mild” and “harsh” experimental conditions. Considering the physiological fold determined by NMR as a benchmark, Figure 6a illustrates the root mean square deviation (RMSD) of the protein backbone along the 500 ns of the MD for the four production runs, i.e. performed on the non-cross-linked and cross-linked cytochrome c thermalized at both 300 K (0 CL_m and 8 CL_m) and 600 K (0 CL_h and 8 CL_h). Results highlight similar RMSD trends comprised between 2 Å and 2.5 Å for all 0 CL_m, 8 CL_m and 8 CL_h, whereas a shift toward twice larger values is observed for 0 CL_h. These data corroborate two aspects evidenced by ion mobility measurements. First, under mild conditions, the cross-linked and the native non-cross-linked folds adopt similar conformations, as further highlighted by the matching superposition of their respective tertiary structures


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achieved after 500 ns (Figure 6b). Second, the cross-linked fold displays higher conformational stability and retains most of the native fold features when harsh experimental conditions are used. This last aspect is highlighted by Figure 6c and 6d showing the superpositions of the tertiary structure of the physiological fold derived from NMR measurements with these respectively achieved for 0 CL_h and 8 CL_h after 500 ns. While numerous identical features relative to the number and alignment of α-helices are shared between the physiological fold and 8 CL_h, the superposition with 0 CL_h highlights substantial discrepancies materialized by diverse losses and/or displacement of secondary structures. This last aspect accounts for unfolding events occurring with harsh experimental conditions in the absence of linkers.

Figure 6. Molecular dynamics simulations performed for 500 ns at 300 K on the non-crosslinked and 8-time cross-linked cytochrome c (z = +8) after thermalization at 300 K (0 CL_m and 8 CL_m) and at 600 K (0 CL_h and 8 CL_h). (a) RMSDs of the protein backbone along



the MD runs considering the physiological fold determined by NMR as a benchmark. (b) Superimpositions of the tertiary structures of the native non-cross-linked fold (0 CL_m) and cross-linked fold (8 CL_m) generated under mild experimental conditions. (c-d) Superimpositions of the tertiary structures of the physiological fold derived from NMR measurements with (c) 0 CL_h and (d) 8 CL_h.

CONCLUSION Our study exemplifies the sometimes-delicate task of probing native-relevant folds using mass spectrometry, particularly on small proteins associated with flexible gas-phase conformational landscapes. By minimizing the transmission voltages, selecting low charge states and spraying from physiological solvent, we were able to achieve compact conformations whose CCSs only exceed the neutral solution benchmark value CCSNMR by 5%. However, when deviating from ideal operating conditions, either by increasing the transmission voltage, spraying from denaturing solvent or lengthening the trapping times, this CCS gap becomes larger after structural unfolding materialized by losses and/or displacements of secondary structures, as highlighted by MD simulations. In view of offering sustained conformational stability and limiting unfolding scenarios that may occur when dealing with non-ideal instrumental settings, the implementation of covalent cross-linkers appears as a promising solution and offers several advantages. First, our circular dichroism measurements reveal unaffected contents in secondary structures after CL additions in solution, as such corroborating previous studies that evidenced their structurally non-intrusive nature.30 Second, data monitored from CSD, IM-MS and CIU experiments all converge toward the preservation of folded compact conformations throughout the entire spraying process, gas-phase settling up to 300 ms and activation stages present along the ion transport. Third, based on the CCS distributions and MD simulations, these conformations


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appear faithful to the native fold achievable with the non-cross-linked species using mild conditions. As they constitute other stable products of any cross-linking reaction, the conformational effect induced by mono-linkers (ML) was also assessed. Doing so, we were able to highlight the preservation of compact conformers belonging to zone A when using an intermediate trap bias voltage of 60 V on mono-linked products of the z = +7 ions of cytochrome c. However, their effect on z = +8 mainly resulted in a redistribution of the populations in favor of the more compact conformer B-C (Supporting Information, Figure S8). These observations suggest that ML can act like non-covalent CL, due to the interactions of the inactivated carboxyl group with polar functions in the protein. This scaffold is yet more prone to fail upon substantial unfolding stress, as encountered with stronger electrostatic repulsions at higher charge states. Further developments and optimizations are still to be performed to more soundly consider cross-linking as an accomplished methodology for structural mass spectrometry. First, a reduction in the accessible primary amine content ensuing from lysine-specific cross-linking results in lower ESI-MS signal intensities, in comparison to non-cross-linked samples. In the same vein, although the dominant charge states are identical in the CSDs monitored before and after extensive cross-linking, the obstruction of lysine residues is expected to induce discrepancies in the way the charges are effectively distributed between non-cross-linked and cross-linked conformers. Relocated into the framework of CID or spectroscopic experiments with labile linkers, these variances are expected to result in different fragmentation patterns. The use of alternative cross-linkers that retain the accessibility of amines for protonation or that target other chemical functions should offer possible solutions.66 Next, our results achieved on the z = +8 ions of cytochrome c show that extensive cross-linking is accompanied by a concomitant broadening of the corresponding IM peak width. This feature, attributed to the



formation of increasing numbers of isobaric conformers, decreases the IM resolution power and may impede refined structural interpretations, especially for small models. Moreover, the handling of cross-linkers is not yet integrated in most molecular dynamic software suites so that additional optimizations and assumptions need to be carried out for adequate force field parametrization. From a methodological point of view, the conformational robustness of cross-linked proteins in a wide array of experimental conditions should make them ideal mobility calibrants for instrumentations relying on traveling wave (TWIMS) or trapping (TIMS)40 IM cell. A caseby-case assessment may, however, be necessary as some charge states display broader IM peaks. Additionally, the compaction effect resulting from cross-linking could be exploited as a biologically-relevant probe to gauge the energetic harshness experienced by molecular ions in IM-MS experiments: the extensively cross-linked species constitutes an immutable reference while the conformation of the non-cross-linked species unfolds depending on the conditions. Based on our results evidencing the adequacy of cross-linkers for the preservation of compact physiologically-relevant folds on isolated proteins, and considering recent works acknowledging their application for multiprotein complexes,27 we likely foresee this strategy as an efficient approach for tomorrow’s structural MS. This should essentially target applications where classic native MS is pushed at its limits, either because of the nature of the analytes, e.g. floppy systems with low interconversion energy barriers between conformers, or because of non-ideal instrumental settings, e.g. long trapping times required for action spectroscopy or higher transmission voltages required to improve sensitivity. Its application to probe specific systems associated with highly dynamic tertiary structures like intrinsically disordered proteins (IDPs)67 or certain amyloids68 is also worth investigating as recent studies highlighted good agreement between the distance constraints issued from cross-linking and structural data issued form small-angle X-ray scattering.69,70 Altogether, these achievements


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will contribute to enhance our general understanding of protein structures and will bring additional resources for the establishment of always more accurate biomolecular models.

ASSOCIATED CONTENT Supporting Information Table S1 Synapt G2-Si HDMS (Waters) - Instrument default setup, Table S2 timsTOF (Bruker) - Instrument default setup, Table S3 Synapt G2-Si HDMS (Waters) – List of calibrants, Figure S1 Synapt G2-Si HDMS (Waters) - Ion mobility calibration, Figure S2 Representation of the heme group and BS3 cross-linker and force field parametrization, Figure S3 CL/ML products for β-lactoglobulin (z = +9) and myoglobin (z = +9), Figure S4 CSDs of (non-)cross-linked β-lactoglobulin and myoglobin, Figure S5 TWCCSN2He distributions of βlactoglobulin (z = +9) and myoglobin (z = +9), Figure S6 Positions of the BS3 linkers on the starting geometry of cytochrome c used for MD, Table S4 List of the reported BS3 cross-linked residues for cytochrome c, Figure S7 Positions of the electric charges on the initial geometry of cytochrome c used for MD, Table S5 List of the positions of the electric charges on the initial geometry of cytochrome c used for MD and Figure S8

TW

CCSN2He distributions of

cytochrome c bearing from 0 ML to 3 ML. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS The authors thank Dr. M. Götze for StavroX support and Dr. D. B. Lima for SIM-XL support. E.H. thanks L. Trzpiot, N. Rosière, and N. Tanteliarisoa Haingo for their support in samples preparations, F. Baumans and C. Delvaux for helpful discussions regarding quantification methods, CD data treatment and gas-phase charge repartition. H.G. acknowledges the support of the H2020 FET open project COPAC 766563 for funding and LPCT laboratory at the University of Lorraine for access to computing resources. 25 ACS Paragon Plus Environment


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TABLE OF CONTENTS GRAPHIC (for Table of Contents Only)


Page 34 of 40

100

1 linker 1786 1788

200x molar excess BS3 tR = 2 h R.I. (%)

1825

1805 1808

1800

1830

1820

1848

1840

1867 1869

1854

1872 1864

1860

1851

1845

CLn-3+ML3 1854

1874

1880

0

1900

m/z

1842 1940 1844 1846 1848 1850 1852 1980 1854 1856 1858 1960 20001860 18622020

m/z

2040

8 linkers 1928

7 linkers 1906 1909 6 linkers 1887

1904

1911

1926

1889 1892

5 linkers 1864 1867

1800

1820

1840

1860

1880

1900

1931 1934

1884 1914 1923

1920

1940

1960 9 linkers

m/z

1980

2000

2020

2040

1980

2000

2020

2040

1948

(c)

0 1780

CLn-2+ML2

CLn

5 linkers 1845 1851

(b)

0 1780 100

3 linkers 1828 2 linkers

0 1780

100x molar excess BS3 tR = 30 mins R.I. (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

(a)

CLn-1+ML1

Analytical Chemistry 100

1848

R.I. (%)

40x molar excess BS3 tR = 2 mins R.I. (%)

100

(d)

4 linkers

Page 35 of 40

1950

8 linkers 1926 1926 1931

1946

1953

1943 ACS Paragon Plus Environment 1923 7 linkers 1904 1906

1800

1820

1840

1860

1880

1900

1920

1940

1960

m/z

(a)

20000

[θ]MRE (deg.cm2.dmol-1)

15000

Physiological solvent (highly cross-linked) Analytical Chemistry Page 36 of 40 Physiological solvent (non-cross-linked)

10000

[θ]MRE (deg.cm2.dmol-1)

1 5000 2 3 0 4 -5000 5 6 -10000 7 -15000 8 9 -20000 200 220 240 260 10 wavelength (nm) 11 (c) Physiological 12 20000 (b) Physiological Denaturing Denaturing 13 14 10000 15 0 16 17 -10000 18 -20000 ACS 220 Paragon Plus260 Environment 19 200 240 200 220 240 260 wavelength (nm) wavelength (nm) 20 Non-cross-linked sample 21 Highly cross-linked sample

Physiological solvent

Denaturing solvent

Page 37 of 40 Analytical Chemistry (a) +14 100 100 +7

1000

1500 2000 m/z +8

2500

R.I. (%)

+7

1000

1500 m/z

2000

0 500

+8 +7 1000

100

R.I. (%)

2500

0 500

+12

2000

2500

+8 +7

1000

100

+8

1500 m/z

+7

1500 m/z +8

2000

2500

+7

R.I. (%)

R.I. (%)

8 - 9 CL

3 - 5 CL

1 2 3 0 500 (b) 4 100 5 6 7 8 0 9 500 (c) 10100 11 12 13 14 0 15 500

R.I. (%)

R.I. (%)

No linker

+8

ACS Paragon Plus Environment 1000

1500 m/z

2000

2500

0 500

1000

1500 m/z

2000

2500

(a)

z = +7

(c) Trap bias = 35 V (d) Trap bias = 60V Analytical A B-C DChemistry E A B-C D E

(e) Trap bias = 100V Page 38DofE40 A B-C

1500 1000

CCSN2→He (Å²)

1500

3 CL

CCSN2→He (Å²)

2000 1500

5 CL

1000 500

z = +8

CCSN2→He (Å²)

(f) Trap bias = 35 V A B-C D

10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00

CCSN2→He (Å²)

TW

2500

CCSN2→He (Å²)

10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00

9 CL 40 50 60 70 80 90 100 Trap bias (V)

10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00

TW

CCSN2→He (Å²)

TW

(g) Trap bias = 60V

E

A B-C D

TW

(h) Trap bias = 100V

E

A B-C D

E

2000 1500 1000

0 CL

500 40 50 60 70 80 90 100 2500

CCSN2→He (Å²)

1 CL

2000 1500 2 CL

1000 500 40 50 60 70 80 90 100

3 CL

2500 2000 5 CL

1500 1000 500

ACS Paragon Plus Environment CCSN2→He (Å²)

TW

Physiological solvent

10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00

9 CL

40 50 60 70 80 90 100 Trap bias (V)

10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00

9 CL

500 40 50 60 70 80 90 100

TW

No linker

2 CL

1000

TW

2 CL

1 CL

2000

CCSN2→He (Å²) TW

9 CL

0 CL

500 40 50 60 70 80 90 100

TW

2 CL

TW

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 (b) 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00

No linker

CCSN2→He (Å²)

2000

CCSN2→He (Å²)

TW

Denaturing solvent

CCSN2→He (Å²)

TW

CCSNMR benchmark

Trapping = 50 ms (b) Trapping = 100 ms (c) Trapping = 300 ms (a) Page 39 of 40 Analytical Chemistry A B-C D

F

A B-C D

E

F

A B-C D

E

F

CCSN2 (Å²)

TIMS

CCSN2 (Å²)

TIMS

12 0 14 0 00 16 0 18 0 0 20 0 00 22 0 24 0 0 26 0 0 28 0 0 30 0 00

12 0 14 0 0 16 0 0 18 0 0 20 0 0 22 0 0 24 0 0 26 0 0 28 0 0 30 0 00

ACS Paragon Plus Environment

12 0 14 0 0 16 0 0 18 0 0 20 0 0 22 0 0 24 0 00 26 0 28 0 0 30 0 00

1 2 3 4 0 CL 5 6 7 8 9 1 CL 10 11 12 13 14 2 CL 15 16 17 18 19 3 CL 20 21 22 23 24 5 CL 25 26 27 28 29 9 CL 30 31 32

E

CCSN2 (Å²)

TIMS

(a)

5.0


4.5

Page 40 of 40

RMSD (Å)

4.0

1 3.5 2 3.0 3 2.5 4 2.0 5 1.5 6 0 7 8 9 10 11 12 13 14 15 16 17 (c) 18 19 20 21 22 23 24 25

100

200

300

500

400

Time (ns) 0 CL_m

0 CL_h

8 CL_m

(b)

8 CL_h 0 CL_m 8 CL_m

(d)

ACS Paragon Plus Environment NMR benchmark

0 CL_h

8 CL_h

Covalent Cross-Linking as an Enabler for Structural Mass

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