Article pubs.acs.org/ac
Polymer Topology Revealed by Ion Mobility Coupled with Mass Spectrometry Denis Morsa,† Thomas Defize,‡ Dominique Dehareng,§ Christine Jérôme,‡ and Edwin De Pauw*,† †
Mass Spectrometry Laboratory, University of Liege, B6c Sart-Tilman, B-4000 Liege, Belgium Center for Education and Research on Macromolecules, University of Liege, B6a Sart-Tilman, B-4000 Liege, Belgium § Center for Protein Engineering, University of Liege, B6a Sart-Tilman, B-4000 Liege, Belgium ‡
S Supporting Information *
ABSTRACT: Hyperbranched and star shaped polymers have raised tremendous interest because of their unusual structural and photochemical properties, which provide them potent applications in various domains, namely in the biomedical field. In this context, the development of adequate tools aiming to probe particular three-dimensional features of such polymers is of crucial importance. In this present work, ion mobility coupled with mass spectrometry was used to experimentally derive structural information related to cationized linear and star shaped poly-εcaprolactones as a function of their charge state and chain length. Two major conformations were observed and identified using theoretical modeling: (1) near spherical conformations whose sizes are invariant with the polymer topology for long and lightly charged chains and (2) elongated conformations whose sizes vary with the polymer topology for short and highly charged chains. These conformations were further confirmed by collisional activation experiments based on the ejection thresholds of the coordinated cations that vary according to the elongation amplitude of the polymer chains. Finally, a comparison between solution and gas-phase conformations highlights a compaction of the structure with a loss of specific chain arrangements during the ionization and desolvation steps of the electrospray process, fueling the long-time debated question related to the preservation of the analyte structure during the transfer into the mass spectrometer.
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also been employed for conformational investigations of multicharged linear chains of poly(ethylene glycol) (PEG)18−20 and polylactide (PLA)21 produced by electrospray (ESI).22 The authors reported progressive transitions from spherical to elongated conformations as the polymer chains shorten and the electrostatic repulsions increase. The corresponding 3D structures were assigned by matching experimental and theoretically modeled ccs.14,23,24 In this present work, we analyzed the conformations of the linear and three star-branched poly-ε-caprolactones (PCL) in the gas phase using IM-MS with several objectives in mind. The first one is to probe specific structural properties that are dependent on the polymer topology and check the adequacy of IM-MS to resolve the four topoisomers. The second one is to probe the stability of the different conformations previously identified using collisional activation and to investigate the fate of charged vibrationally excited polymer chains in the gas phase. In the last part, we probed the structural evolution of a polymer chain following the ionization and desolvation steps of
ranched polymers such as dendrimers, hyperbranched or star shaped polymers raise a tremendous interest in macromolecular chemistry because of their attractive properties.1,2 These polymers exhibit not only interesting rheological and mechanical properties but also a lower melt viscosity than their linear counterparts, allowing processing at a lower temperature.3 This point is of particular interest for lowstability polymers such as polylactones, which have attracted increasing attention because of their biodegradability and biocompatibility properties required for biomedicine applications.4,5 Separation and characterization of branched polymers usually rely on size exclusion chromatography coupled with a static light scattering detector.6,7 This technique allows for the determination of the mean-square radius of gyration (Rg) of a polymer chain,8 which is related to its volume and namely depends on its topology.9 Recent studies show that ion mobility coupled with mass spectrometry (IM-MS) is another valuable tool to study polymer samples.10,11 This technique consists of the separation of ions according to their gas-phase size and shape,12−14 gathered under the term collision cross section (ccs) Ω.15 In this context, topology-based separations of cyclic and branched polymers have been reported.16,17 IM-MS has © 2014 American Chemical Society
Received: June 18, 2014 Accepted: August 31, 2014 Published: September 4, 2014 9693
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the ESI process by comparing its collision cross section Ω in the gas phase using IM-MS and its radius of gyration (Rg) in solution using light-scattering measurements. This analysis is of particular interest to understand the fate of sprayed ions and to assess the possibility to retrieve solution conformations using data acquired in the gas phase. Exporting this debate in the biology field, the preservation of the native structure of protein complexes during its transfer into the mass spectrometer is a crucial step for native-MS investigations.25 Although a memory of the solution-phase structure seems to be retained through the ESI process,26,27 recent works suggest a series of partial structural rearrangements occurring within very short time scales28−30 and a definitive idea on this subject is still to be formulated.
Ion Mobility Experiments. Polymer samples were diluted in a 50/50 mix of tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) saturated with potassium bromide (KBr). They were injected at 10−5 mol/L using electrospray in a firstgeneration SYNAPT instrument (Waters, Manchester, U.K.) equipped with a traveling-wave ion mobility cell (TWIMS).34,35 This cell is fitted between a quadrupole and a time-of-flight (TOF) mass analyzer. The following parameters were used for each samples: capillary voltage =3 kV, sampling cone =30 V, extraction cone =3 V, source temperature =110 °C, desolvation temperature =200 °C, cone gas flow =22 L/h, desolvation gas flow =100 L/h, trap/transfer CE =4 V, bias =13 V, IMS wave height =15 V, IMS wave speed =600 m/s and IMS pressure =0.356 mbar. Acquired data was processed using Waters Masslynx 4.1 and Driftscope 2.1 software. Arrival time distribution (ATD) peaks were fitted using PeakFit v. 4.11 by Systat Software. Collision Cross Section (ccs) Calibration. The relation between the drift time td and the ion mobility K, related to its collision cross section Ω, is not yet fully elucidated in travelingwave ion mobility cells.35 We therefore performed a calibration based on the methodology described by Ruotolo et al.24 using reported ccs values for bradykinin,36 myoglobine,37 ubiquitine,38 cytochrome C,39 a tryptic digest of bovine serum albumin BSA40 and polylactides.21 Calibration curve along with a list of selected calibrants are reported in the Supporting Information (Figure S-2 and Table S-3). Computational Chemistry and Theoretical ccs Calculations. Reported structures were determined at the molecular dynamics (MD) level using the general amber force field (GAFF)41 coupled with calculation of net electric charges using AM1-BCC,42 as implemented in YASARA modeling program (http://www.yasara.org). For each reported system, three random starting conformations were built and followed for at least 50 ns at two distinct temperatures: 300 and 600 K. Structures generated all along the dynamics were systematically picked up and their collision cross sections Ω were calculated based on the exact hard sphere scattering (EHSS) method implemented in the EHSSrot software.43 The selection of candidates was established on the basis of a match between the experimental and the theoretical ccs.
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EXPERIMENTAL SECTION Chemicals. ε-Caprolactone (εCL) (Janssen Chimica), toluene (Chem-Lab) and 1,4-butanediol (Aldrich) were dried over calcium hydride for 24 h at room temperature and distilled under reduced pressure just before use. Pentaerythritol (SigmaAldrich), dipentaerythritol (Sigma-Aldrich) and tripentaerythritol (Sigma-Aldrich) were dried by three azeotropic distillations of toluene just before polymerization. Tin octoate (SnOct2, Aldrich), and heptane (Chem-Lab) were used as received. Synthesis of PCLs. Ring opening polymerization was performed at 110 °C in bulk under inert atmosphere using alcohol as the initiator and tin octoate as a catalyst.31−33 1,4Butanediol, pentaerythritol, dipentaerythritol and tripentaerythritol were used to respectively obtain the linear, 4-arm star, 6arm star and 8-arm star shaped PCLs. The conversion of the monomer was followed by 1H NMR and stopped when quantitative by cooling the reactor. The resulting product was dissolved in toluene and purified by precipitation in heptane under stirring. The PCL was then recovered by filtration and dried under vacuum. Characterization of PCLs. Molecular parameters of polymers were determined by size exclusion chromatography (SEC) in dimethylformamide (DMF) containing some LiBr (0.025 M) at 55 °C (flow rate: 1 mL/min) using a Waters 600 liquid chromatograph equipped with a 410 refractive index detector and styragel HR columns (polystyrene calibration). Absolute molecular weight was determined using a multi-angle laser light scattering (MALLS) detector (λ = 658 nm). Data were processed with the Astra V software (Wyatt Technology). 1 H NMR spectra were recorded at 400 MHz in the FT mode with a Bruker AN 400 apparatus at 25 °C, using CDCl3 as solvent and internal standard. Obtained results are given in the Supporting Information, Table S-1. Radii of gyration were determined using size exclusion chromatography (SEC) in tetrahydrofuran (THF) at 45 °C at a flow rate of 0.7 mL/min with Viscotek 305 TDA liquid chromatograph. The PL gel 5 μm (104, 103 and 100 Å) columns were calibrated with Malvern polyCAL TDS-PS-NB polystyrene standard. The GPC system is equipped with 7° low angle laser light scattering (LALLS) detector and 90° right angle laser light scattering detector (RALLS), which were both set with 670 nm laser light source, differential refractometer and viscometer in series. The samples were filtered through Nylon Acrodisc filters with a pore size of 0.2 μm before being injected into the GPC systems. The scattering signals were collected and analyzed with OmniSEC software.
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RESULTS AND DISCUSSION Arrival Time Distributions of PCLs. Figure 1 shows the 2D arrival time distribution (ATD) obtained for the linear PCL. ATDs obtained for the 4-arm star, 6-arm star and 8-arm star shaped PCLs present similar features (Supporting Information, Figure S-4) and are discussed in details further. The x-axis and y-axis respectively correspond to the mass-to-charge m/z ratios and to the ion mobility drift times td. This last quantity depends on the ion mobility K,35 which is governed by the charge-tocollision cross section ratio z/Ω, as given by the Mason− Shamp eq 1:44 K=
1/2 3 e ⎛ 2π ⎞ z ⎜ ⎟ 16 N ⎝ μkBT ⎠ Ω
(1)
where e is the elementary charge, kB is the Boltzmann constant, T is the gas temperature, N is the gas number density and μ is the reduced mass of the collision partners. Four different charge states z, from +1 to +4, were generated by coordination of potassium ions (K+)45 onto the polymer chains using ESI22 and are accordingly labeled in Figure 1. Due 9694
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chains of decreasing length (from right to left in Figure 1), a steady evolution of the drift times td with decreasing m/z ratios is first observed for all charge states z. This situation is correlated with the retention of a unique molecular shape of gradually decreasing size as the polymer chains shortens. Next, sequential abrupt increases of the drift time td appear for gradually less charged species, starting from z = +4 to z = +2. This situation is correlated with drastic conformational rearrangements that promote the formation of elongated molecular shapes whose both collision cross sections Ω and ion mobility drift times td are higher. Similar observations were reported for studies based on polyethylene glycol (PEG) coordinating NH4+19 and Cs+,18 and polylactide (PLA) coordinating Na+.21 The authors assumed that a polymer chain adopts a roughly spherical shape providing a sufficient screening of the repulsive interactions occurring between coordinated cations. As the chain shortens, this screening is not sufficient anymore and the chain adopts an elongated shape promoted by a greater distance between the cations. Ude et al. explain this structural transition using an analogy with the Rayleigh limit for a liquid drop.20 The Rayleigh’s criterion46 correlates the critical mass m*, at which structural instabilities appear, with the physical properties of the drop. It is given by eq 2:
Figure 1. 2D ATD distribution for the linear PCL. Chains coordinating up to four K+ were observed. The distributions corresponding to the different charge states are split at high masses but interconvert at low masses because of conformational rearrangements imparted to electrostatic repulsions.
to the polydispersity of the polymer sample, each charge state hosts a mass peak distribution associated with polymer chains of distinct degrees of polymerization (DP).11 Considering PCL
Figure 2. Collision cross section Ω of polymer chains as a function of the degree of polymerization (DP) for the four charge states of the linear (a), 4-arm star (b), 6-arm star (c) and 8-arm star (d) topoisomers. At high DP, all distributions collapse onto a common curve associated with nearly spherical conformations, regardless of the polymer topology. When the chain length decreases, deviations from the curve arise and are correlated with electrostatically driven elongations of the polymer chains. 9695
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Analytical Chemistry e 2ρ 1 m* = 2 γε0 48π z
Article
(2)
Where ρ is the density, γ is the surface tension and ε0 is the electric permittivity of the vacuum. As the limit m*/z2 is constant for a given polymer, the structural transitions appear at a higher m/z ratio as the charge state z increases. This explains the trends observed in the left part of Figure 1. Comparison between Different Topologies. To facilitate the comparison between ATDs obtained for the four PCL topoisomers, m/z ratios and drift times td were respectively converted to degrees of polymerization (DP) and to collision cross sections (ccs) Ω and reported as a function of each other. Generated data are displayed in Figure 2 a, b, c and d for respectively the linear, 4-arm star, 6-arm star and 8-arm star shaped PCLs. Note that some points are missing for the distributions corresponding to z = +4 due to a superposition with other charge states. For each topology, two distinct situations arise and can be correlated with observations highlighted in Figure 1. For PCL chains of high DP, the four distributions corresponding to the different charge states collapse onto a common curve (gray line). This curve was fitted for each topoisomers using the power equation given by eq 3:
Ω = A(DP)B
Figure 3. Collision cross section expansion ΔΩ as a function of the degree of polymerization (DP). For a same degree of polymerization, the ccs expansion gets higher as the charge state increases and the branching ratio decreases. Three distinct structural families, labeled F1, F2 and F3, are formed during the chain elongation process.
and large ccs fluctuations. Lesser sloped segments are characteristic of stable conformations whose size is not very sensitive to fluctuations of DP. On the basis of the analysis of the slopes for z = +4, three stable conformational families, labeled F1, F2 and F3, were spotted and highlighted in Figure 3. F1 is only observable for highly branched PCLs, F2 is present for the four topoisomers and F3 is only observable for the linear PCL. These families respectively arise for ccs expansions of ∼120, ∼220 and ∼320 Å2. For z = +4, the linear 36-mer belongs to the F3 family whereas its branched topoisomers belong to the F2 family. This shows that topoisomers of identical length may adopt different conformations and confirms that their separation using IM-MS is feasible. Molecular Modeling. 50 ns molecular dynamics (MD) simulations were used to derive structural information related to the linear, 4-arm star and 6-arm star shaped PCLs. We first modeled a 36-mer coordinating two K+ for each topology. On the basis of Figure 2, they all should adopt similar nearly spherical shapes. Our theoretical results agree with the experiments and demonstrate the formation of slightly ellipsoidal structures of similar collision cross sections Ω for all topoisomers (Supporting Information, Figure S-5). The difference between the experimental and the theoretical ccs is lower than 2% and is comprised within the experimental errors. We then performed calculations on chains coordinating four K+, both for the 36-mers and for a linear 48-mer. Obtained conformations are illustrated in Figure 4 and look very similar to those previously reported for elongated PEG chains.19 Their pattern consists of a large spherical or ellipsoidal bead coordinating most of K+ from which extends a 5-monomer long linker ending with a smaller polymer globule typically comprising 8 to 9 monomers and coordinating a lone K+. The structures of the branched 36-mers and of the linear 48-mer all belong to the F2 family and share a similar 3−1 K+ distribution: three K+ are aggregated in the large polymeric bead and the fourth one is isolated in the ending globule. The large bead shows a slight elongation for the branched 36-mers whereas it remains roughly spherical for the linear 48-mer. The structure of the linear 36-mer belongs to the F3 family and displays a distinct 2−1−1 K+ distribution as one K+ from the large bead is now located on the linker. The origin of these structural differences may be found in the stability of the large polymer
(3)
Where A and B are the fitting parameters. Similar values of these parameters were obtained resulting in A̅ = 48 ± 1 and B̅ = 0.71 ± 0.01. This last value is in good adequacy with B = 2/3, which is awaited for a perfect totally dense spherical shape.24 This result further supports the idea that long polymer chains adopt nearly spherical conformations, regardless of their topologies. For chains of lower DP, deviations from the curve occur and are correlated with structural transitions leading from a nearly spherical shape to an elongated shape of respective higher ccs. Although the ccs of the nearly spherical conformers are independent of the topology, differences can be found for shorter elongated chains. To better visualize them, we reported the collision cross section expansion ΔΩ as a function of the degree of polymerization for z = +2, z = +3 and z = +4 of each topoisomer in Figure 3. ΔΩ is defined as the difference between the experimental ccs and the corresponding spherical ccs calculated using the fitted curve equation. Structural deviations appear at similar DPs for all topologies depending on the charge state z and correspond to a critical Rayleigh ratio m*/z2 of ∼500 Da. However, once the elongation process started, the ccs expansion ΔΩ becomes dependent on the topology and decreases with the branching ratio. This effect can be correlated with a diminution of the chain flexibility which restricts the elongation of highly branched polymers. Topoisomers of identical and rather short lengths thus adopt elongated conformations of different size which are distinguishable with the resolving power of IM-MS. The analysis of Figure 3 also reveals that parts of the curves associated with the elongation process display variable slopes over the studied DP domain. This phenomenon is especially visible for z = +4 and can be correlated with distinct stretching responses to a modification of the chain length.20 The steepest parts of the curves are associated with structures whose size is very sensitive to little fluctuations of DP. Small variations in the chain length therefore induce drastic structural rearrangements 9696
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signal intensity arising from z = +4 over the total signal intensity of the MS spectrum. As illustrated, the ejection threshold increases with the chain length and is shifted from 32 V for the 32-mer to 95 V for the 70-mer for 50% of dissociation. This namely results from two effects. First, the energy of collision Ecoll depends on the mass of the ion mion, as given by eq 4:47 Ecoll = E k,ion,lab
mgas mgas + m ion
= q·V ·
mgas mgas + m ion
(4)
where Ek,ion,lab is the kinetic energy of the ion in the lab frame, mgas and mion are respectively the masses of the gas and of the ion, q is the charge of the ion and V is the accelerative voltage. This energy is defined as the maximum amount of energy transferred from kinetic motion to internal degrees of freedom upon a single inelastic collision and is proportional to the total amount of transferred energy during the entire activation process. Second, an intrinsic kinetic shift results from the transferred energy redistribution in all available internal degrees of freedom whose amount nDOF increases with the mass of the ion.48 To overcome the dependence on the ion mass and therefore on the chain length, we normalized the survival curves both according to Ecoll and nDOF (Figure 6b). The discrepancies in the ejection thresholds are now only imparted to differences in the stability of the K+ on the polymer chain. After normalization, the ejection thresholds increase with the relative size of the structures, starting from the most compact spherical shape (70-mer), going through the partially elongated F2 chains (55-mer and 48-mer) and ending with the most elongated F3 chains (36-mer and 32-mer). This ranking is correlated with a diminution of the long-range repulsions occurring between the coordinated K+ as the chain elongation drives them apart. To further support the experimental results, we compared the energy of a modeled 36-mer coordinating four K+ with its stable stretched homologue. It appears that the average distance between cations increases from 15 to 35 Å when going from a spherical shape to an elongated shape (Supporting Information, Figure S-7). This spacing induces a decrease of the Coulomb energy ΔECoulomb of 84 kJ/mol, a subsequent stabilization of coordinated K+ and an increase of the corresponding ejection threshold. This theory is sustained by Figure 6c showing a gathering of the survival curves after normalization according to QΩ, which is defined as the ratio between the experimental ccs and the corresponding spherical ccs. Assuming of a proportional evolution between the ccs and the charge density ρq, this gathering proves that the electrostatic repulsions play the key role in the ranking of the ejection thresholds reported in Figure 6b. Chain Conformations in Solution and in the Gas Phase. Structural reorganizations consecutive to the ionization and desolvation processes occurring during the transfer of polymer chains into the mass spectrometer were probed for the four topoisomers (linear, 4-arm star, 6-arm star and 8-arm star) of similar average length (DP = 65). PCL chains were first solubilized in tetrahydrofuran (THF), which promotes the formation of solvent-swollen random coil conformations.49 Their weight-average radius of gyration (Rgw) values were determined using light-scattering measurements.7 Dimethyl sulfoxide (DMSO) and KBr were then added, and the resulting cationized chains were sprayed into the mass spectrometer. As previously showed, the quadruply charged 65-mers adopt a compact nearly spherical shape in the gas phase, regardless of
Figure 4. MD structures of the linear 36-mer (a), 4-arm star 36-mer (b), 6-arm star 36-mer (c) and linear 48-mer (d). The linear 36-mer belongs to the F3 structural family which displays a distinct 2−1−1 K+ distribution whereas the three others species belong to the F2 family characterized by a 3−1 K+ distribution.
bead. This one comprises 23 and 37 monomers for, respectively, a 36-mer and a 48-mer. Figure 3 shows that only the triply charged 37-mer is able to adopt a nearly spherical shape whereas the triply charged 23-mers adopt an elongated shape. The linear 36-mer is therefore stabilized by isolation of a second K+, resulting in the formation of a stable doubly charged spherical large bead whereas its branched counterparts are stabilized by an elongation of the large bead, which increases the average distance between the coordinated K+. Collisional Activation as a Structural Probe. To test the relative stability of the different conformations previously observed, we performed collisional activations on linear PCL chains selected in the quadrupole and we monitored their MS profile as a function of increasing accelerative voltages. Five chains comprising 32, 36, 48, 55, and 70 monomers and coordinating four K+ were studied. On the basis of Figure 3, the 70-mer adopts a spherical shape, the 55-mer and 48-mer both belong to the F2 family characterized by a 3−1 K+ distribution and the 36-mer and 32-mer belong to the F3 family characterized by a 2−1−1 K+ distribution. MS spectra of the 36-mer and 48-mer are shown in Figure 5a,b, respectively; additional spectra are shown in the Supporting Information (Figure S-6). As illustrated, an increase of the accelerative voltage induces sequential ejections of one K+ with a concomitant reduction of the charge state. However, no covalent fragmentation of the polymer chain is observed which is awaited based on the lower dissociation energy of an ion−dipole interaction (∼5 kcal/mol) compared to a covalent bond (∼100 kcal/mol). For each of the five selected chains, we monitored the survival yield (SY) for the +4 charge state as a function of the accelerative voltage (Figure 6a). SY is equal to the ratio of the 9697
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Figure 5. MS spectra of the quadrupole-selected 36-mer (a) and 48-mer (b) coordinating four K+ recorded as functions of the accelerative voltage. Collisional activation induces sequential ejections of K+ with no covalent fragmentation of the polymer chain. The ejection threshold is shifted to higher voltages as the chain length increases. Peaks marked with an asterisk are impurities.
chains that are able to coordinate all cations in a same large bead. The size of the spheres is not dependent on the polymer topology, making separation of topoisomers using IM-MS impossible within this structural domain. Short and highly charged chains adopt elongated conformations because of electrostatic repulsions occurring between coordinated cations. Both the experimental results and the MD simulations revealed that different elongated conformations characterized by particular distributions of K+ on the polymer chain are formed depending on the degree of polymerization (DP), on the charge state z and on the branching ratio. This last dependence can be imparted to differences in the flexibility of the polymer chains and makes separation of topoisomers using IM-MS feasible within this structural domain. Collisional activation performed on selected cationized polymer chains induce sequential ejections of the coordinated K+. The ejection threshold is dependent on the PCL chain conformation and, after normalizations according to both the energy of collision Ecoll and the number of vibrational normal modes nDOF, increases with the amplitude of the elongation. This is correlated with an increase of the average distance between the coordinated K+ as the chain elongates, which induces a diminution of the electrostatic repulsions. Altogether,
their topology. Their collision cross sections were measured and the corresponding radii were calculated based on the equation of a circle area. Obtained results are displayed in Table 1. The determined Rgw values are in good adequacy with results reported by Huanga et al.49 and exhibit an expected dependence on the polymer topology.50 The transfer of the polymer chains into the mass spectrometer induces a diminution of the radius, witnessing a compaction of the structure. This observation could be imparted to the evaporation of the inflating solvent from the random coil, leading to collapsing empty cavities and ultimately to compact structures in the gas phase. Because of their rather dense nature, the radius of these structures is not dependent anymore on the chain topology. This shows that the discrimination of PCL topoisomers using IM-MS requires the presence of sufficient electrostatic repulsions to induce the formation of topologydependent elongated conformations.
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CONCLUSION Our results show that cationized PCL chains adopt either spherical or elongated conformations in the gas phase. Spherical conformations arise for long and lightly charged 9698
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Finally, we investigated the structural fate of PCL chains when transferred from a solution to the gas phase by electrospray (ESI). In solution, the polymer adopts a random-coil conformation whose size varies according to the topology. The desolvation process induces a contraction of the structure imparted to a partial collapse of the solvent-swollen cavities. This ultimately leads to the formation of compact spheres in the gas phase whose size is not dependent anymore on the polymer topology. This result shows that some specific structural arrangements are lost during the vaporization process and that the resulting gas-phase conformation does not strictly reflect all the details of the initial solution conformation. Exporting this idea to the biology field, desolvation of proteins and nucleic acids could also lead to structural compactions and even to partial rearrangements of the native structures.30 This last step would strongly depend on the intramolecular interactions, especially on hydrogen bonding, and their ability to maintain the shape of the molecule intact. Recent studies based on the coupling of capillary electrophoresis (CE) with IM-MS51,52 could help in comparing the size of molecules in solution and in the gas phase and bring some answers to this largely debated question.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E. De Pauw. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the F.R.S.-FNRS for financial support. Professor Bernard Leyh, Professor Philippe Lecomte and Dr. Raphael Riva are acknowledged for helpful discussions.
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Figure 6. Survival curves corresponding to the extinction of z = +4 after ejection of one K+ recorded as functions of the accelerative voltage (a). Survival curves after normalization according to the energy of collision Ecoll and according to the number of vibrational normal modes nDOF (b). Survival curves after normalization according to the ccs ratio QΩ (c).
Table 1. Comparison between the Radii of Solvent-Swollen Random Coils in Solution (THF) and Compact Sphere in the Gas Phase for 65 Monomer Long PCL Chains Rgw (nm) solution (THF) linear 4-arm star 6-arm star 8-arm star
3.03 2.81 2.67 2.52
R (nm) gas phase 1.81 1.83 1.83 1.78
± ± ± ±
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0.02 0.02 0.01 0.02
these results show that collisional activation is a relevant tool to study the conformations of PCL in the gas phase and to further support IM-MS data. 9699
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(14) López, A.; Tarragó, T.; Vilaseca, M.; Giralt, E. New J. Chem. 2013, 37, 1283. (15) Shvartsburg, A. A. Differential Ion Mobility Spectrometry: Nonlinear Ion Transport and Fundamentals of FAIMS; CRC Press Inc: Boca Raton, FL, 2008; pp 1−54. (16) Anthony, P.; Gies, M. K.; John, A.; McLean; Hercules, D. M. Macromolecules 2008, 41, 8299−8301. (17) Hoskins, J. N.; Trimpin, S.; Grayson, S. M. Macromolecules 2011, 44, 6915−6918. (18) Trimpin, S.; Plasencia, M.; Isailovic, D.; Clemmer, D. E. Anal. Chem. 2007, 79, 7965−7974. (19) Larriba, C.; de la Mora, J. F. J. Phys. Chem. B 2012, 116, 593− 598. (20) Ude, S.; Fernández de la Mora, J.; Thomson, B. A. J. Am. Chem. Soc. 2004, 126, 12184−12190. (21) De Winter, J.; Lemaur, V.; Ballivian, R.; Chirot, F.; Coulembier, O.; Antoine, R.; Lemoine, J.; Cornil, J.; Dubois, P.; Dugourd, P.; Gerbaux, P. Chemistry 2011, 17, 9738−9745. (22) O’Connor, P.; McLafferty, F. J. Am. Chem. Soc. 1995, 117, 12826−12831. (23) Saikusa, K.; Fuchigami, S.; Takahashi, K.; Asano, Y.; Nagadoi, A.; Tachiwana, H.; Kurumizaka, H.; Ikeguchi, M.; Nishimura, Y.; Akashi, S. Anal. Chem. 2013, 85, 4165−4171. (24) Ruotolo, B. T.; Benesch, J. L.; Sandercock, A. M.; Hyung, S. J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139−1152. (25) Heck, A. J. Nat. Methods 2008, 5, 927−933. (26) Ruotolo, B. T.; Robinson, C. V. Curr. Opin. Chem. Biol. 2006, 10, 402−408. (27) Wanga, F.; Freitasa, M. A.; Marshall, A. G.; Sykes, B. D. Int. J. Mass Spectrom. 1999, 192, 319−325. (28) Hogan, C. J., Jr.; Ruotolo, B. T.; Robinson, C. V.; Fernandez de la Mora, J. J. Phys. Chem. B 2011, 115, 3614−3621. (29) Breuker, K.; Jin, M.; Han, X.; Jiang, H.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 2008, 19, 1045−1053. (30) Breuker, K.; McLafferty, F. W. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18145−18152. (31) Kricheldorf, H. R.; Kreiser-Saunders, I.; Boettcher, C. Polymer 1995, 36, 1253. (32) Kowalski, A.; Duda, A.; Penczek, S. Macromol. Rapid Commun. 1998, 19, 567−572. (33) Storey, R. F.; Taylor, A. E. J. Macromol. Sci. Pure Appl. Chem. 1998, 35, 723. (34) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.; Bateman, R. H. Rapid Commun. Mass Spectrom. 2004, 18, 2401−2414. (35) Shvartsburg, A. A.; Smith, R. D. Anal. Chem. 2008, 80, 9689− 9699. (36) Counterman, A. E.; Valentine, S. J.; Srebalus, C. A.; Henderson, S. C.; Hoaglund, C. S.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1998, 9, 743−759. (37) Shelimov, K. B.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2987−2994. (38) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1997, 8, 954−961. (39) Chen, Y.-L.; Collings, B. A.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1997, 8, 681−687. (40) Bush, M. F.; Campuzano, I. D. G.; Robinson, C. V. Anal. Chem. 2012, 84, 7124−7130. (41) Wang, J.; Wolf, R.; Caldwell, J.; Kollman, P.; Case, D. J. Comput. Chem. 2004, 25, 1157−1174. (42) Jakalian, A.; Jack, D.; Bayly, C. J. Comput. Chem. 2002, 23, 1623−1641. (43) Shvartsburg, A. A.; Mashkevich, S. V.; Baker, E. S.; Smith, R. D. J. Phys. Chem. A 2007, 111, 2002−2010. (44) Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases; Wiley: New York, 1988. (45) Jasieczek, C. B.; Buzy, A.; Haddleton, D. M.; Jennings, K. R. Rapid Commun. Mass Spectrom. 1996, 10, 509−514. (46) Rayleigh, L. Philos. Mag. 1882, 14, 184−186.
(47) McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1992, 3, 599−614. (48) Lifshitz, C. Eur. J. Mass Spectrom. 2002, 8, 85−98. (49) Huanga, Y.; Xua, Z.; Huangb, Y.; Mab, D.; Yangc, J.; Maysc, J. W. Int. J. Polym. Anal. Charact. 2003, 8, 383−394. (50) Zimm, B. H.; Stockmayer, W. H. J. Chem. Phys. 1949, 17, 1301. (51) Jiang, X. M.; Svensmark, B.; Deng, L. H. Adv. Mater. Res. 2010, 160−162, 1531−1534. (52) Far, J.; Kune, C.; Delvaux, C.; Burmistrova, A.; Morsa, D.; Pauw, E. D. In 32nd Informal Meeting on Mass Spectrometry, Balatonszarszo, Hungary, May 11−14, 2014.
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Polymer Topology Revealed by Ion Mobility Coupled with Mass Spectrometry Denis Morsa,† Thomas Defize,‡ Dominique Dehareng,§ Christine Jérôme,‡ and Edwin De Pauw*,† †
Mass Spectrometry Laboratory, University of Liege, B6c Sart-Tilman, B-4000 Liege, Belgium Center for Education and Research on Macromolecules, University of Liege, B6a Sart-Tilman, B-4000 Liege, Belgium § Center for Protein Engineering, University of Liege, B6a Sart-Tilman, B-4000 Liege, Belgium ‡
S Supporting Information *
ABSTRACT: Hyperbranched and star shaped polymers have raised tremendous interest because of their unusual structural and photochemical properties, which provide them potent applications in various domains, namely in the biomedical field. In this context, the development of adequate tools aiming to probe particular three-dimensional features of such polymers is of crucial importance. In this present work, ion mobility coupled with mass spectrometry was used to experimentally derive structural information related to cationized linear and star shaped poly-εcaprolactones as a function of their charge state and chain length. Two major conformations were observed and identified using theoretical modeling: (1) near spherical conformations whose sizes are invariant with the polymer topology for long and lightly charged chains and (2) elongated conformations whose sizes vary with the polymer topology for short and highly charged chains. These conformations were further confirmed by collisional activation experiments based on the ejection thresholds of the coordinated cations that vary according to the elongation amplitude of the polymer chains. Finally, a comparison between solution and gas-phase conformations highlights a compaction of the structure with a loss of specific chain arrangements during the ionization and desolvation steps of the electrospray process, fueling the long-time debated question related to the preservation of the analyte structure during the transfer into the mass spectrometer.
B
also been employed for conformational investigations of multicharged linear chains of poly(ethylene glycol) (PEG)18−20 and polylactide (PLA)21 produced by electrospray (ESI).22 The authors reported progressive transitions from spherical to elongated conformations as the polymer chains shorten and the electrostatic repulsions increase. The corresponding 3D structures were assigned by matching experimental and theoretically modeled ccs.14,23,24 In this present work, we analyzed the conformations of the linear and three star-branched poly-ε-caprolactones (PCL) in the gas phase using IM-MS with several objectives in mind. The first one is to probe specific structural properties that are dependent on the polymer topology and check the adequacy of IM-MS to resolve the four topoisomers. The second one is to probe the stability of the different conformations previously identified using collisional activation and to investigate the fate of charged vibrationally excited polymer chains in the gas phase. In the last part, we probed the structural evolution of a polymer chain following the ionization and desolvation steps of
ranched polymers such as dendrimers, hyperbranched or star shaped polymers raise a tremendous interest in macromolecular chemistry because of their attractive properties.1,2 These polymers exhibit not only interesting rheological and mechanical properties but also a lower melt viscosity than their linear counterparts, allowing processing at a lower temperature.3 This point is of particular interest for lowstability polymers such as polylactones, which have attracted increasing attention because of their biodegradability and biocompatibility properties required for biomedicine applications.4,5 Separation and characterization of branched polymers usually rely on size exclusion chromatography coupled with a static light scattering detector.6,7 This technique allows for the determination of the mean-square radius of gyration (Rg) of a polymer chain,8 which is related to its volume and namely depends on its topology.9 Recent studies show that ion mobility coupled with mass spectrometry (IM-MS) is another valuable tool to study polymer samples.10,11 This technique consists of the separation of ions according to their gas-phase size and shape,12−14 gathered under the term collision cross section (ccs) Ω.15 In this context, topology-based separations of cyclic and branched polymers have been reported.16,17 IM-MS has © 2014 American Chemical Society
Received: June 18, 2014 Accepted: August 31, 2014 Published: September 4, 2014 9693
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the ESI process by comparing its collision cross section Ω in the gas phase using IM-MS and its radius of gyration (Rg) in solution using light-scattering measurements. This analysis is of particular interest to understand the fate of sprayed ions and to assess the possibility to retrieve solution conformations using data acquired in the gas phase. Exporting this debate in the biology field, the preservation of the native structure of protein complexes during its transfer into the mass spectrometer is a crucial step for native-MS investigations.25 Although a memory of the solution-phase structure seems to be retained through the ESI process,26,27 recent works suggest a series of partial structural rearrangements occurring within very short time scales28−30 and a definitive idea on this subject is still to be formulated.
Ion Mobility Experiments. Polymer samples were diluted in a 50/50 mix of tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) saturated with potassium bromide (KBr). They were injected at 10−5 mol/L using electrospray in a firstgeneration SYNAPT instrument (Waters, Manchester, U.K.) equipped with a traveling-wave ion mobility cell (TWIMS).34,35 This cell is fitted between a quadrupole and a time-of-flight (TOF) mass analyzer. The following parameters were used for each samples: capillary voltage =3 kV, sampling cone =30 V, extraction cone =3 V, source temperature =110 °C, desolvation temperature =200 °C, cone gas flow =22 L/h, desolvation gas flow =100 L/h, trap/transfer CE =4 V, bias =13 V, IMS wave height =15 V, IMS wave speed =600 m/s and IMS pressure =0.356 mbar. Acquired data was processed using Waters Masslynx 4.1 and Driftscope 2.1 software. Arrival time distribution (ATD) peaks were fitted using PeakFit v. 4.11 by Systat Software. Collision Cross Section (ccs) Calibration. The relation between the drift time td and the ion mobility K, related to its collision cross section Ω, is not yet fully elucidated in travelingwave ion mobility cells.35 We therefore performed a calibration based on the methodology described by Ruotolo et al.24 using reported ccs values for bradykinin,36 myoglobine,37 ubiquitine,38 cytochrome C,39 a tryptic digest of bovine serum albumin BSA40 and polylactides.21 Calibration curve along with a list of selected calibrants are reported in the Supporting Information (Figure S-2 and Table S-3). Computational Chemistry and Theoretical ccs Calculations. Reported structures were determined at the molecular dynamics (MD) level using the general amber force field (GAFF)41 coupled with calculation of net electric charges using AM1-BCC,42 as implemented in YASARA modeling program (http://www.yasara.org). For each reported system, three random starting conformations were built and followed for at least 50 ns at two distinct temperatures: 300 and 600 K. Structures generated all along the dynamics were systematically picked up and their collision cross sections Ω were calculated based on the exact hard sphere scattering (EHSS) method implemented in the EHSSrot software.43 The selection of candidates was established on the basis of a match between the experimental and the theoretical ccs.
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EXPERIMENTAL SECTION Chemicals. ε-Caprolactone (εCL) (Janssen Chimica), toluene (Chem-Lab) and 1,4-butanediol (Aldrich) were dried over calcium hydride for 24 h at room temperature and distilled under reduced pressure just before use. Pentaerythritol (SigmaAldrich), dipentaerythritol (Sigma-Aldrich) and tripentaerythritol (Sigma-Aldrich) were dried by three azeotropic distillations of toluene just before polymerization. Tin octoate (SnOct2, Aldrich), and heptane (Chem-Lab) were used as received. Synthesis of PCLs. Ring opening polymerization was performed at 110 °C in bulk under inert atmosphere using alcohol as the initiator and tin octoate as a catalyst.31−33 1,4Butanediol, pentaerythritol, dipentaerythritol and tripentaerythritol were used to respectively obtain the linear, 4-arm star, 6arm star and 8-arm star shaped PCLs. The conversion of the monomer was followed by 1H NMR and stopped when quantitative by cooling the reactor. The resulting product was dissolved in toluene and purified by precipitation in heptane under stirring. The PCL was then recovered by filtration and dried under vacuum. Characterization of PCLs. Molecular parameters of polymers were determined by size exclusion chromatography (SEC) in dimethylformamide (DMF) containing some LiBr (0.025 M) at 55 °C (flow rate: 1 mL/min) using a Waters 600 liquid chromatograph equipped with a 410 refractive index detector and styragel HR columns (polystyrene calibration). Absolute molecular weight was determined using a multi-angle laser light scattering (MALLS) detector (λ = 658 nm). Data were processed with the Astra V software (Wyatt Technology). 1 H NMR spectra were recorded at 400 MHz in the FT mode with a Bruker AN 400 apparatus at 25 °C, using CDCl3 as solvent and internal standard. Obtained results are given in the Supporting Information, Table S-1. Radii of gyration were determined using size exclusion chromatography (SEC) in tetrahydrofuran (THF) at 45 °C at a flow rate of 0.7 mL/min with Viscotek 305 TDA liquid chromatograph. The PL gel 5 μm (104, 103 and 100 Å) columns were calibrated with Malvern polyCAL TDS-PS-NB polystyrene standard. The GPC system is equipped with 7° low angle laser light scattering (LALLS) detector and 90° right angle laser light scattering detector (RALLS), which were both set with 670 nm laser light source, differential refractometer and viscometer in series. The samples were filtered through Nylon Acrodisc filters with a pore size of 0.2 μm before being injected into the GPC systems. The scattering signals were collected and analyzed with OmniSEC software.
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RESULTS AND DISCUSSION Arrival Time Distributions of PCLs. Figure 1 shows the 2D arrival time distribution (ATD) obtained for the linear PCL. ATDs obtained for the 4-arm star, 6-arm star and 8-arm star shaped PCLs present similar features (Supporting Information, Figure S-4) and are discussed in details further. The x-axis and y-axis respectively correspond to the mass-to-charge m/z ratios and to the ion mobility drift times td. This last quantity depends on the ion mobility K,35 which is governed by the charge-tocollision cross section ratio z/Ω, as given by the Mason− Shamp eq 1:44 K=
1/2 3 e ⎛ 2π ⎞ z ⎜ ⎟ 16 N ⎝ μkBT ⎠ Ω
(1)
where e is the elementary charge, kB is the Boltzmann constant, T is the gas temperature, N is the gas number density and μ is the reduced mass of the collision partners. Four different charge states z, from +1 to +4, were generated by coordination of potassium ions (K+)45 onto the polymer chains using ESI22 and are accordingly labeled in Figure 1. Due 9694
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chains of decreasing length (from right to left in Figure 1), a steady evolution of the drift times td with decreasing m/z ratios is first observed for all charge states z. This situation is correlated with the retention of a unique molecular shape of gradually decreasing size as the polymer chains shortens. Next, sequential abrupt increases of the drift time td appear for gradually less charged species, starting from z = +4 to z = +2. This situation is correlated with drastic conformational rearrangements that promote the formation of elongated molecular shapes whose both collision cross sections Ω and ion mobility drift times td are higher. Similar observations were reported for studies based on polyethylene glycol (PEG) coordinating NH4+19 and Cs+,18 and polylactide (PLA) coordinating Na+.21 The authors assumed that a polymer chain adopts a roughly spherical shape providing a sufficient screening of the repulsive interactions occurring between coordinated cations. As the chain shortens, this screening is not sufficient anymore and the chain adopts an elongated shape promoted by a greater distance between the cations. Ude et al. explain this structural transition using an analogy with the Rayleigh limit for a liquid drop.20 The Rayleigh’s criterion46 correlates the critical mass m*, at which structural instabilities appear, with the physical properties of the drop. It is given by eq 2:
Figure 1. 2D ATD distribution for the linear PCL. Chains coordinating up to four K+ were observed. The distributions corresponding to the different charge states are split at high masses but interconvert at low masses because of conformational rearrangements imparted to electrostatic repulsions.
to the polydispersity of the polymer sample, each charge state hosts a mass peak distribution associated with polymer chains of distinct degrees of polymerization (DP).11 Considering PCL
Figure 2. Collision cross section Ω of polymer chains as a function of the degree of polymerization (DP) for the four charge states of the linear (a), 4-arm star (b), 6-arm star (c) and 8-arm star (d) topoisomers. At high DP, all distributions collapse onto a common curve associated with nearly spherical conformations, regardless of the polymer topology. When the chain length decreases, deviations from the curve arise and are correlated with electrostatically driven elongations of the polymer chains. 9695
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(2)
Where ρ is the density, γ is the surface tension and ε0 is the electric permittivity of the vacuum. As the limit m*/z2 is constant for a given polymer, the structural transitions appear at a higher m/z ratio as the charge state z increases. This explains the trends observed in the left part of Figure 1. Comparison between Different Topologies. To facilitate the comparison between ATDs obtained for the four PCL topoisomers, m/z ratios and drift times td were respectively converted to degrees of polymerization (DP) and to collision cross sections (ccs) Ω and reported as a function of each other. Generated data are displayed in Figure 2 a, b, c and d for respectively the linear, 4-arm star, 6-arm star and 8-arm star shaped PCLs. Note that some points are missing for the distributions corresponding to z = +4 due to a superposition with other charge states. For each topology, two distinct situations arise and can be correlated with observations highlighted in Figure 1. For PCL chains of high DP, the four distributions corresponding to the different charge states collapse onto a common curve (gray line). This curve was fitted for each topoisomers using the power equation given by eq 3:
Ω = A(DP)B
Figure 3. Collision cross section expansion ΔΩ as a function of the degree of polymerization (DP). For a same degree of polymerization, the ccs expansion gets higher as the charge state increases and the branching ratio decreases. Three distinct structural families, labeled F1, F2 and F3, are formed during the chain elongation process.
and large ccs fluctuations. Lesser sloped segments are characteristic of stable conformations whose size is not very sensitive to fluctuations of DP. On the basis of the analysis of the slopes for z = +4, three stable conformational families, labeled F1, F2 and F3, were spotted and highlighted in Figure 3. F1 is only observable for highly branched PCLs, F2 is present for the four topoisomers and F3 is only observable for the linear PCL. These families respectively arise for ccs expansions of ∼120, ∼220 and ∼320 Å2. For z = +4, the linear 36-mer belongs to the F3 family whereas its branched topoisomers belong to the F2 family. This shows that topoisomers of identical length may adopt different conformations and confirms that their separation using IM-MS is feasible. Molecular Modeling. 50 ns molecular dynamics (MD) simulations were used to derive structural information related to the linear, 4-arm star and 6-arm star shaped PCLs. We first modeled a 36-mer coordinating two K+ for each topology. On the basis of Figure 2, they all should adopt similar nearly spherical shapes. Our theoretical results agree with the experiments and demonstrate the formation of slightly ellipsoidal structures of similar collision cross sections Ω for all topoisomers (Supporting Information, Figure S-5). The difference between the experimental and the theoretical ccs is lower than 2% and is comprised within the experimental errors. We then performed calculations on chains coordinating four K+, both for the 36-mers and for a linear 48-mer. Obtained conformations are illustrated in Figure 4 and look very similar to those previously reported for elongated PEG chains.19 Their pattern consists of a large spherical or ellipsoidal bead coordinating most of K+ from which extends a 5-monomer long linker ending with a smaller polymer globule typically comprising 8 to 9 monomers and coordinating a lone K+. The structures of the branched 36-mers and of the linear 48-mer all belong to the F2 family and share a similar 3−1 K+ distribution: three K+ are aggregated in the large polymeric bead and the fourth one is isolated in the ending globule. The large bead shows a slight elongation for the branched 36-mers whereas it remains roughly spherical for the linear 48-mer. The structure of the linear 36-mer belongs to the F3 family and displays a distinct 2−1−1 K+ distribution as one K+ from the large bead is now located on the linker. The origin of these structural differences may be found in the stability of the large polymer
(3)
Where A and B are the fitting parameters. Similar values of these parameters were obtained resulting in A̅ = 48 ± 1 and B̅ = 0.71 ± 0.01. This last value is in good adequacy with B = 2/3, which is awaited for a perfect totally dense spherical shape.24 This result further supports the idea that long polymer chains adopt nearly spherical conformations, regardless of their topologies. For chains of lower DP, deviations from the curve occur and are correlated with structural transitions leading from a nearly spherical shape to an elongated shape of respective higher ccs. Although the ccs of the nearly spherical conformers are independent of the topology, differences can be found for shorter elongated chains. To better visualize them, we reported the collision cross section expansion ΔΩ as a function of the degree of polymerization for z = +2, z = +3 and z = +4 of each topoisomer in Figure 3. ΔΩ is defined as the difference between the experimental ccs and the corresponding spherical ccs calculated using the fitted curve equation. Structural deviations appear at similar DPs for all topologies depending on the charge state z and correspond to a critical Rayleigh ratio m*/z2 of ∼500 Da. However, once the elongation process started, the ccs expansion ΔΩ becomes dependent on the topology and decreases with the branching ratio. This effect can be correlated with a diminution of the chain flexibility which restricts the elongation of highly branched polymers. Topoisomers of identical and rather short lengths thus adopt elongated conformations of different size which are distinguishable with the resolving power of IM-MS. The analysis of Figure 3 also reveals that parts of the curves associated with the elongation process display variable slopes over the studied DP domain. This phenomenon is especially visible for z = +4 and can be correlated with distinct stretching responses to a modification of the chain length.20 The steepest parts of the curves are associated with structures whose size is very sensitive to little fluctuations of DP. Small variations in the chain length therefore induce drastic structural rearrangements 9696
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signal intensity arising from z = +4 over the total signal intensity of the MS spectrum. As illustrated, the ejection threshold increases with the chain length and is shifted from 32 V for the 32-mer to 95 V for the 70-mer for 50% of dissociation. This namely results from two effects. First, the energy of collision Ecoll depends on the mass of the ion mion, as given by eq 4:47 Ecoll = E k,ion,lab
mgas mgas + m ion
= q·V ·
mgas mgas + m ion
(4)
where Ek,ion,lab is the kinetic energy of the ion in the lab frame, mgas and mion are respectively the masses of the gas and of the ion, q is the charge of the ion and V is the accelerative voltage. This energy is defined as the maximum amount of energy transferred from kinetic motion to internal degrees of freedom upon a single inelastic collision and is proportional to the total amount of transferred energy during the entire activation process. Second, an intrinsic kinetic shift results from the transferred energy redistribution in all available internal degrees of freedom whose amount nDOF increases with the mass of the ion.48 To overcome the dependence on the ion mass and therefore on the chain length, we normalized the survival curves both according to Ecoll and nDOF (Figure 6b). The discrepancies in the ejection thresholds are now only imparted to differences in the stability of the K+ on the polymer chain. After normalization, the ejection thresholds increase with the relative size of the structures, starting from the most compact spherical shape (70-mer), going through the partially elongated F2 chains (55-mer and 48-mer) and ending with the most elongated F3 chains (36-mer and 32-mer). This ranking is correlated with a diminution of the long-range repulsions occurring between the coordinated K+ as the chain elongation drives them apart. To further support the experimental results, we compared the energy of a modeled 36-mer coordinating four K+ with its stable stretched homologue. It appears that the average distance between cations increases from 15 to 35 Å when going from a spherical shape to an elongated shape (Supporting Information, Figure S-7). This spacing induces a decrease of the Coulomb energy ΔECoulomb of 84 kJ/mol, a subsequent stabilization of coordinated K+ and an increase of the corresponding ejection threshold. This theory is sustained by Figure 6c showing a gathering of the survival curves after normalization according to QΩ, which is defined as the ratio between the experimental ccs and the corresponding spherical ccs. Assuming of a proportional evolution between the ccs and the charge density ρq, this gathering proves that the electrostatic repulsions play the key role in the ranking of the ejection thresholds reported in Figure 6b. Chain Conformations in Solution and in the Gas Phase. Structural reorganizations consecutive to the ionization and desolvation processes occurring during the transfer of polymer chains into the mass spectrometer were probed for the four topoisomers (linear, 4-arm star, 6-arm star and 8-arm star) of similar average length (DP = 65). PCL chains were first solubilized in tetrahydrofuran (THF), which promotes the formation of solvent-swollen random coil conformations.49 Their weight-average radius of gyration (Rgw) values were determined using light-scattering measurements.7 Dimethyl sulfoxide (DMSO) and KBr were then added, and the resulting cationized chains were sprayed into the mass spectrometer. As previously showed, the quadruply charged 65-mers adopt a compact nearly spherical shape in the gas phase, regardless of
Figure 4. MD structures of the linear 36-mer (a), 4-arm star 36-mer (b), 6-arm star 36-mer (c) and linear 48-mer (d). The linear 36-mer belongs to the F3 structural family which displays a distinct 2−1−1 K+ distribution whereas the three others species belong to the F2 family characterized by a 3−1 K+ distribution.
bead. This one comprises 23 and 37 monomers for, respectively, a 36-mer and a 48-mer. Figure 3 shows that only the triply charged 37-mer is able to adopt a nearly spherical shape whereas the triply charged 23-mers adopt an elongated shape. The linear 36-mer is therefore stabilized by isolation of a second K+, resulting in the formation of a stable doubly charged spherical large bead whereas its branched counterparts are stabilized by an elongation of the large bead, which increases the average distance between the coordinated K+. Collisional Activation as a Structural Probe. To test the relative stability of the different conformations previously observed, we performed collisional activations on linear PCL chains selected in the quadrupole and we monitored their MS profile as a function of increasing accelerative voltages. Five chains comprising 32, 36, 48, 55, and 70 monomers and coordinating four K+ were studied. On the basis of Figure 3, the 70-mer adopts a spherical shape, the 55-mer and 48-mer both belong to the F2 family characterized by a 3−1 K+ distribution and the 36-mer and 32-mer belong to the F3 family characterized by a 2−1−1 K+ distribution. MS spectra of the 36-mer and 48-mer are shown in Figure 5a,b, respectively; additional spectra are shown in the Supporting Information (Figure S-6). As illustrated, an increase of the accelerative voltage induces sequential ejections of one K+ with a concomitant reduction of the charge state. However, no covalent fragmentation of the polymer chain is observed which is awaited based on the lower dissociation energy of an ion−dipole interaction (∼5 kcal/mol) compared to a covalent bond (∼100 kcal/mol). For each of the five selected chains, we monitored the survival yield (SY) for the +4 charge state as a function of the accelerative voltage (Figure 6a). SY is equal to the ratio of the 9697
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Figure 5. MS spectra of the quadrupole-selected 36-mer (a) and 48-mer (b) coordinating four K+ recorded as functions of the accelerative voltage. Collisional activation induces sequential ejections of K+ with no covalent fragmentation of the polymer chain. The ejection threshold is shifted to higher voltages as the chain length increases. Peaks marked with an asterisk are impurities.
chains that are able to coordinate all cations in a same large bead. The size of the spheres is not dependent on the polymer topology, making separation of topoisomers using IM-MS impossible within this structural domain. Short and highly charged chains adopt elongated conformations because of electrostatic repulsions occurring between coordinated cations. Both the experimental results and the MD simulations revealed that different elongated conformations characterized by particular distributions of K+ on the polymer chain are formed depending on the degree of polymerization (DP), on the charge state z and on the branching ratio. This last dependence can be imparted to differences in the flexibility of the polymer chains and makes separation of topoisomers using IM-MS feasible within this structural domain. Collisional activation performed on selected cationized polymer chains induce sequential ejections of the coordinated K+. The ejection threshold is dependent on the PCL chain conformation and, after normalizations according to both the energy of collision Ecoll and the number of vibrational normal modes nDOF, increases with the amplitude of the elongation. This is correlated with an increase of the average distance between the coordinated K+ as the chain elongates, which induces a diminution of the electrostatic repulsions. Altogether,
their topology. Their collision cross sections were measured and the corresponding radii were calculated based on the equation of a circle area. Obtained results are displayed in Table 1. The determined Rgw values are in good adequacy with results reported by Huanga et al.49 and exhibit an expected dependence on the polymer topology.50 The transfer of the polymer chains into the mass spectrometer induces a diminution of the radius, witnessing a compaction of the structure. This observation could be imparted to the evaporation of the inflating solvent from the random coil, leading to collapsing empty cavities and ultimately to compact structures in the gas phase. Because of their rather dense nature, the radius of these structures is not dependent anymore on the chain topology. This shows that the discrimination of PCL topoisomers using IM-MS requires the presence of sufficient electrostatic repulsions to induce the formation of topologydependent elongated conformations.
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CONCLUSION Our results show that cationized PCL chains adopt either spherical or elongated conformations in the gas phase. Spherical conformations arise for long and lightly charged 9698
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Finally, we investigated the structural fate of PCL chains when transferred from a solution to the gas phase by electrospray (ESI). In solution, the polymer adopts a random-coil conformation whose size varies according to the topology. The desolvation process induces a contraction of the structure imparted to a partial collapse of the solvent-swollen cavities. This ultimately leads to the formation of compact spheres in the gas phase whose size is not dependent anymore on the polymer topology. This result shows that some specific structural arrangements are lost during the vaporization process and that the resulting gas-phase conformation does not strictly reflect all the details of the initial solution conformation. Exporting this idea to the biology field, desolvation of proteins and nucleic acids could also lead to structural compactions and even to partial rearrangements of the native structures.30 This last step would strongly depend on the intramolecular interactions, especially on hydrogen bonding, and their ability to maintain the shape of the molecule intact. Recent studies based on the coupling of capillary electrophoresis (CE) with IM-MS51,52 could help in comparing the size of molecules in solution and in the gas phase and bring some answers to this largely debated question.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E. De Pauw. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the F.R.S.-FNRS for financial support. Professor Bernard Leyh, Professor Philippe Lecomte and Dr. Raphael Riva are acknowledged for helpful discussions.
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Figure 6. Survival curves corresponding to the extinction of z = +4 after ejection of one K+ recorded as functions of the accelerative voltage (a). Survival curves after normalization according to the energy of collision Ecoll and according to the number of vibrational normal modes nDOF (b). Survival curves after normalization according to the ccs ratio QΩ (c).
Table 1. Comparison between the Radii of Solvent-Swollen Random Coils in Solution (THF) and Compact Sphere in the Gas Phase for 65 Monomer Long PCL Chains Rgw (nm) solution (THF) linear 4-arm star 6-arm star 8-arm star
3.03 2.81 2.67 2.52
R (nm) gas phase 1.81 1.83 1.83 1.78
± ± ± ±
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