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Tandem Differential Mobility Analysis-Mass Spectrometry Reveals Partial Gas-Phase Collapse of the GroEL Complex Christopher J. Hogan, Jr.,†,‡,|| Brandon T. Ruotolo,§ Carol V. Robinson,^ and Juan Fernandez de la Mora*,† †
Department of Mechanical Engineering, Yale University, New Haven, Connecticut, United States SEADM, Boecillo, Spain § Department of Chemistry, University of Michigan, Ann Arbor, Michigan, United States ^ Department of Chemistry, University of Oxford, Oxford, England, United Kingdom ‡
bS Supporting Information ABSTRACT: A parallel-plate differential mobility analyzer and a time-of-flight mass spectrometer (DMA-MS) are used in series to measure true mobility in dry atmospheric pressure air for massresolved electrosprayed GroEL tetradecamers (14-mers; ∼800 kDa). Narrow mobility peaks are found (2.6-2.9% fwhm); hence, precise mobilities can be obtained for these ions without collisional activation, just following their generation by electrospray ionization. In contrast to previous studies, two conformers are found with mobilities (Z) differing by ∼5% at charge state z ∼ 79. By extrapolating to small z, a common mobility/charge ratio Z0/z = 0.0117 cm2 V-1 s-1 is found for both conformers. When interpreted as if the GroEL ion surface were smooth and the gas molecule-protein collisions were perfectly elastic and specular, this mobility yields an experimental collision cross section, Ω, 11% smaller than in an earlier measurement, and close to the cross section, AC,crystal, expected for the crystal structure (determined by a geometric approximation). However, the similarity between Ω and AC,crystal does not imply a coincidence between the native and gas-phase structures. The nonideal nature of protein-gas molecule collisions introduces a drag enhancement factor, ξ = 1.36, with which the true cross section AC is related to Ω via AC = Ω/ ξ. Therefore, AC for GroEL 14-mer ions determined by DMA measurements is 0.69AC,crystal. The factor 1.36 used here is based on the experimental Stokes-Millikan equation, as well as on prior and new numerical modeling accounting for multiple scattering events via exact hard-sphere scattering calculations. Therefore, we conclude that the gas-phase structure of the GroEL complex as electrosprayed is substantially more compact than the corresponding X-ray crystal structure.
’ INTRODUCTION Measurement of large proteins and protein complex ions by tandem ion mobility spectrometry-mass spectrometry (IMSMS) enables investigation of gas-phase protein ion structure.1-5 In most studies of protein complexes by IMS-MS, the transition from solution to gas phase is brought about via electrospray ionization, in which multiply charged gas-phase ions are produced from dissolved protein complexes by the evaporation of multiply charged water drops.6-9 The similarities and differences between solution and gas-phase protein structures and the ability to use such gas-phase data to retrieve solution-phase relevant information are, therefore, of great interest. Many structural features of dissolved proteins are retained upon the drying of electrospray drops.10 On a global level, analogues of acid denaturation are observed for charged gas-phase ions;11,12 noncovalent bonds between proteins and ligands are often retained in the transition;13 electrosprayed viruses14,15 can remain viable and enzymes16 can remain active; and a ring-structured protein complex can remain intact in the absence of bulk solvent.1 r 2011 American Chemical Society
Conversely, on a more localized structural level, recent work by Breuker, McLafferty, and co-workers17-19 suggests that proteins undergo a series of structural changes on nanosecond and millisecond time scales, which could complicate precise structural analysis of protein tertiary and secondary structure by IMS-MS. Further examination of model protein ion systems is clearly necessary to better determine under what experimental conditions and to what extent solution-phase protein structure is retained upon introduction into the gas phase by electrospray ionization. The present study uses mobility data obtained in dry air for the large GroEL tetradecamer (14-mer) complex20 to address two issues relevant to studying the effects of the solution to gas-phase transition on protein ion structure. The first is that extraneous shape changes may arise2 when the ions enter into the vacuum Received: September 25, 2010 Revised: January 29, 2011 Published: March 11, 2011 3614
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The Journal of Physical Chemistry B system of the MS, or are injected into the mobility cell in drift tube21 or T-wave1 based IMS-MS systems. This problem is sidestepped here by performing the mobility measurement with a differential mobility analyzer (DMA, a low-field, linear mobility instrument)22 at atmospheric pressure, prior to any such ion activation. Second, it is often of interest to infer a measure of the ions' size from mobility measurements, such as the ions0 crosssectional area or mobility diameter. Unlike collision cross sections,23-26 these measures can be compared to cross-sectional areas or mobility diameters27,28 determined through alternative techniques.5 Conversion of mobilities into an appropriate measure of ion size should incorporate a reduction factor, ξ (to be referred to as a drag enhancement factor) for the actual mobility relative to that associated with elastic specular collisions with smooth surfaces. A value ξ = 1.36 based on Millikan’s oil drop experiments is ubiquitous in the field of aerosol science,29 not only in air but in a variety of other gases such as He and CO2.30-37 With the drag enhancement factor, mobility measurements have been used to determine the diameter of size standard polystyrene latex spheres as well as a number of inorganic nanoparticles.38-41 In each of these cases, the nanoparticle diameters inferred from measurements are in very good agreement with measurements made using electron microscopy. Furthermore, with drag enhancement accounted for, mobility measurements of known mass ions can be used to infer ion density,27,37,42 allowing for further comparison to alternative measurement techniques (e.g., density measurements of proteins in solution). While cross-sectional areas and mobility diameters can also be inferred for protein ions, Millikan’s ξ is only sparingly cited in studies of gas-phase biomolecules.43 In several cases, inferences of biomolecular ion size have been made without accounting for ξ, and in these instances ion sizes were overpredicted5 and densities underpredicted.44 In the analysis of biomolecular ions, the mobility associated to a given ion structure is more commonly predicted via the exact hard-sphere scattering model (EHSS).26 In the EHSS approach, the ion surface is represented by an assembly of atoms, each treated as a smooth sphere undergoing elastic collisions with specular reflections with the gas. Analysis of prior results with this model shows ξEHSS depending on geometry and ion size, exceeding in some cases ξEHSS = 1.20.45 Calculations for symmetrically growing aggregates of fullerenes yielded an enhancement factor decreasing linearly with curvature and extrapolating at large sizes to 1.3 (Figure 3 of Shvartsburg et al.26), directly comparable to Millikan’s value, evidently also corresponding to large objects.42 Here, we will show through a new EHSS calculation and through the related computations of ref 3 (both yielding ξ = 1.36) that Millikan’s measured drag enhancement factor is valid for GroEL 14-mer ions.
’ EXPERIMENTAL METHODS GroEL Sample Preparation and Electrospray Ionization. GroEL 14-mers were purchased from Sigma-Aldrich (C7688) and purified as described previously.46 For electrospray ionization, the GroEL complexes were dissolved in 100 mM aqueous ammonium acetate at a concentration of several micromoles. Sample solution was driven through a 40 μm inner diameter capillary (360 μm outer diameter, tapered down to ∼60 μm at the outlet, length ∼25 cm) with a backing pressure of 0.05 bar. A silver wire (which does not oxidize or promote any
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electrochemical reactions within the sample vial) was used to apply a high voltage (EMCO high voltage) directly to the sample solution. This high voltage was set to float above the potential applied to the DMA upper electrode and to operate the electrospray source at a constant current (∼200 nA) in the cone-jet mode. Cone-jet operation was verified by visually examining the electrospray capillary outlet with a microscope camera with the electrospray source several centimeters from the DMA-MS inlet. 1.0 L min-1 of dry CO2 was passed through the electrospray source region to aid in maintaining a stable cone-jet electrospray. For DMA-MS measurements, the electrospray source was moved close to the DMA inlet (∼1 mm between the capillary outlet and DMA inlet) and the high voltage applied to the sample solution was reduced accordingly to maintain a constant electrospray current. Differential Mobility Analysis-Mass Spectrometry. The operating principles of differential mobility analyzers have been reviewed previously.22,47-49 DMA-MS was performed as is described elsewhere42,49,50 with parallel-plate DMA P4 (SEADM, Boecillo, Spain) coupled to a QSTAR XL mass spectrometer (MDS Sciex). The DMA was operated with a recirculating sheath flow of air and with a counterflow of air at the DMA inlet (∼0.3 L min-1), which prevented CO2 and uncharged solvent vapor from entering the DMA. The temperature of the DMA was measured as 31 C. For DMA calibration, the voltage required to transmit the cluster ion (tetrahepytlammoniumþ)3(Br-)2 was determined, which has a mobility of approximately 0.538 cm2 V-1 s-1 in air at 31 C.51 With the mobility scale of the DMA calibrated, the mobility of each measured ion was determined from the equation Z¼
Zs V s VDMA
ð1Þ
where Z is the ion mobility, VDMA is the applied voltage in the DMA, Zs is the mobility of the standard, and Vs is the DMA voltage required to transmit the standard ion. The lower DMA electrode containing the ion outlet to the MS was interfaced directly with the lens and skimmer of the mass spectrometer inlet. Following Chernushevich and Thomson,52 an additional sleeve (commercially available from MDS Sciex) was attached to the skimmer of the mass spectrometer to increase the pressure in the quadrupole ion guide. This enhances the thermalization of very large ions and enables their efficient transmission to the detector. Mass spectra were measured using the time-of-flight section of the QSTAR XL in the 1000-35 000 m/z (mass-to-charge ratio) range. Tandem mobility mass spectra were acquired by taking mass spectra with the DMA operating at fixed voltage for a period of 40 s. After complete measurement of a mass spectrum at fixed DMA voltage, the DMA voltage was increased by 10 V, and a new mass spectrum was subsequently measured. Theoretical Calculations of Collision Cross Section from Atomic Coordinates. In addition to estimates of GroEL size based on geometric approximations described in detail elsewhere in this text, we also used the atomic coordinates of GroEL (PDB ID 1SX3) to estimate the collision cross section of the ion using MOBCAL26 (http://www.indiana.edu/30% shift in cross section below the crystal structure value). ðz=ZÞ ¼ ðz=Z0 Þð1 þ bz Þ
ð2aÞ
2
54
Prior measurements of denatured protein ions and polymer ions55 show that z/Z depends upon z in a more complex fashion
than assumed in (2a). However, this is the case only when the proteins suffer a drastic loss of compactness associated to denaturing. Although our data correspond to near-neutral pH solutions and are free from such problems, this precedent suggests that eq 2a should be used prudently. Interestingly, although the two conformers have quite different mobilities, both series extrapolate at zero charge to very much the same value: (Z0/z) = 0.0117 cm2 V-1 s-1 for the more abundant conformer and 0.0116 cm2 V-1 s-1 for the less abundant. With the extrapolation, it seems as if the two conformations are identical in the absence of Coulombic forces, with one being more sensitive to the charge than the other. Previous IMS-MS measurements on GroEL, using both linear-field56 and T-wave instruments,3 have seen substantially smaller z. One of these studies has reported considerable z/Z dependence on charge state,3 but not the other.56 The DMA-MS measurements reported here are unique in that the maximum z expected for a protein with the mass of GroEL is zmax = 71,6 while we see a clear peak at z = 82. The fact that there are two closely related gas-phase conformations of GroEL is also intriguing, as is the finding that both have indistinguishable structures but different sensitivity to charge state. Measured Cross Section of GroEL. The z-corrected mobility Z0 defined in eq 2a may now be used conventionally to define the experimental collision cross section Ω via eq 2b sffiffiffiffiffiffiffiffiffiffiffi Z0 3 e 2πkT ð2bÞ ¼ 16 pΩ mg z where e is the electron charge, k is Boltzmann’s constant, T is the gas temperature, p is the gas pressure, and mg is the gas molecule mass. Ω is often interpreted as the cross-sectional area of the ion. However, as already noted, this definition of eq 2b is strictly applicable only to hard spheres with elastic and specular reflections. For macroscopic objects with rough surfaces, the actual geometrical cross section, AC differs from Ω through a drag enhancement factor ξ: AC ¼ Ω=ξ 2
-1
ð2cÞ
-1
Using Z0/z = 0.0117 cm V s extrapolated from our measurements and the value ξ = 1.36 (previously justified based on both experiment and calculation and discussed further below), direct use of the free-molecule expression 2b gives AC = Ω/ξ = [π(di þ dg)2/4] = 132 nm2, where di is the ion mobility diameter and dg is the gas molecule diameter (∼0.3 nm for air).27,37 The resulting di = 12.68 nm ignores continuum corrections not included in eqs 2a and 2b. These are conventionally accounted for in aerosol studies in the so-called Millikan formula,27,29,31,36 where the ratio of the free-molecule mobility Z0 and the actual mobility ZM (both corrected for polarization effects) for spherical ions as small as those of interest here may be written: di þ dg ZM ¼ Z0 1 þ 0:338 ð3Þ 2λ Τhe continuum correction factor, in parentheses in eq 3, accounts for the finite ratio between the sphere diameter and the gas mean free path λ (2λ ∼ 134 nm in ambient air). It is very close to unity in the free-molecule limit typical of low-pressure drift tubes, as well as with 1 nm ions at atmospheric pressure. For our 3617
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The Journal of Physical Chemistry B
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to the surface) over the surface of the body, treated as macroscopically smooth and hard with global effects of the locally rough surface included in the enhancement factor ξ = 1.36. The approach generalizes eq 2b for spheres to arbitrary convex shapes. The mobility Z may be written in terms of the total surface area (wetted area) of the complex, ATot = π(di þ dg)2 = 4ΑC as !1=2 Ζ 1 e 9πkT ¼ ð5Þ z ξ pATot 8mg
Figure 4. Axial and cross-sectional views of GroEL 14-mers, as well as a image of the geometric approximation used to calculate its electrical mobility.
conditions ZM/Z0 = 1.034, yielding the corrected cross-sectional area and mobility diameter: AC ¼ 137 nm2 ,
di ¼ 12:94 nm
ð4Þ
The continuum correction expression used is valid only for spheres, so its application to a nonspherical object of the same mobility is only an approximation. It should nonetheless decrease the already small error well below 1%. The determined AC =137 nm2 is much below the 244 nm2 previously reported and compared to a projection approximation,3 which was based on the assumption ξ = 1. However, meaningful comparison between the two reported cross-sectional areas requires correcting the prior measurement similarly for charge state influences as well as ξ. Examining the highest charge state inferred collision cross sections (at charge states 53-56 and 68-72) from the T-wave measurements of van Duijn et al.3 as a function of z2 gives an extrapolated zero-charge ΑC value of ∼154 nm2, which is only 11% greater than our zero charge AC of 137 nm2. The even more recent drift tube measurements reported in Bush et al.56 do not reveal a charge state dependence for the inferred GroEL 14-mer ion collision cross sections, in either He or N2 buffer gas. Nonetheless, there is reasonable agreement between drift tube and DMA data. From our measurements of the more abundant GroEL conformer at z = 70 we infer ΑC,z=70 of 155 nm2 without correcting for charge effects. This is only 4% different from the reported ΑC,z=70 of 161 nm2 by Bush et al.57 in N2. The differences between three independent measurements, though small, highlight (1) how ion activation and location of the mobility measurement device relative to the ionization source can alter the structure of a gas-phase ion as well as (2) the effects of charge on a gas-phase ion mobility and structure (polarization and Coulombic stretching) need to be taken into consideration for multiply charged ions. Expected Mobility of Liquid-Phase Structure. Front and side views of the crystal structure of GroEL 14-mer are represented in Figure 4 from the protein databank (file 1KPO). The 14-mer resembles closely a cylinder with a length L ∼14 nm, and a base composed of N = 7 identical triangles of height h ∼ 6.5 nm. The mobility of this cylinder was estimated using a simple algorithm57 that integrates the tensor nn dA (n is the normal
The advantage of this representation is that the same expression derived for spheres applies to objects having more than one axis of rotational symmetry, for which the drag tensor is isotropic.57 Regular polygonal cylinders have only one symmetry axis. However, the drag tensor is also isotropic when the cylinder length is twice the height of the N triangles forming the polygonal base: L = 2h. This geometrical circumstance is very close to being true for GroEL; thus, its drag tensor is almost isotropic, with negligible anisotropy corrections. Therefore, eq 5 provides almost exactly the mobility of GroEL in any of its possible orientations. This result is also important because the calculation of the anisotropy correction involves a proportionality constant not known from Millikan’s work (with isotropic spheres), which is at present computed based on Epstein’s inelastic model.58 The coincidental negligible anisotropy correction for the geometry of GroEL removes the ambiguity associated with this anisotropy constant. As an additional benefit of this circumstance, the computed mobility would apply even if the ion was not sampling all possible orientations, but rather adopted a fixed direction within the DMA. Note, however, that the relevant area is the total wetted area of the ion, not the projected area sometimes used to approximate the cross section of nonspherical ions. For the heptagonal cylinder model of GroEL, ATot is given as eq 6a: ATot ¼ 2NLh tanðπ=NÞ þ Nh2 tanðπ=NÞ
ð6aÞ
Using N = 7, h = 6.5 nm and L = 14 nm, yields ATot = 756 nm2. The finite diameter of the air molecules can be accounted for approximately by increasing L and 2h by 0.3 nm,27 to yield ATot ¼ 790 nm2 ;
AC, crystal ¼ 197 nm2
ð6bÞ
In this model, the open regions on the surface of the GroEL complex are neglected, as is the opening on the axis, which has a diameter of ∼5 nm. We now consider the possible effect of the cavity on ion mobility. Most gas molecules entering through one end of the cavity will have directions sufficiently away from the axis such that they will not leave directly through the other end, but will undergo at least one internal collision. Most of these air molecules will undergo not just one, but many collisions with the internal walls. Accordingly, they will be thermalized yielding their full momentum to the protein, with no additional net force being applied as they eventually leave with symmetric velocity distributions through both openings. Ignoring small associated anisotropy effects, the main effect of the openings is therefore that they act as if ξ were unity instead of 1.36 (ξ = 1 for collisions with hard spheres both when the reflection is specular, and when the collision transmits only the full momentum of the collider). This reduces the effective wetted area from ξATot into ξ(ATot - Aop) þ Aop, where Aop is the total area of the opening. The drag therefore decreases by a factor 1 - (1 - ξ-1)Aop/ATot. Using 3618
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The Journal of Physical Chemistry B ξ = 1.36, a cavity diameter of 5 nm, and ATot = 790 nm2, this factor differs from unity by only 0.42%. The effect would be even smaller (0.37%) if we reduced the cavity diameter by the finite size of the air molecules. The additional reduction in drag associated with molecules flying through the cavity without collisions is even smaller, given the small probability for such narrow range of directions. These openings can therefore introduce only minimal shifts in the computed Z/z. Two alternative calculations of the surface area of GroEL based on the projection approximation PA to the cross section, ΩPA and on protein databank files will be discussed at the end of this paper. They yield 217 and 220 nm2,3 11% larger than (6b). This ambiguity in AC,crystal is relatively minor given that the computed native wetted area, (6b) is 1.44 times larger than the measured area (4), and comparably larger than the crosssectional areas (AC rather than Ω) of Van Duijn et al.3 and Bush et al.56 Therefore, the gas phase GroEL 14-mer is actually observed to be more compact than expected based on multiple experimental data sets. It is important to note that the magnitude of compression observed for the GroEL 14-mer is the largest currently recorded for any protein or multiprotein complex in the gas phase. We have recently analyzed by DMA-MS a number of proteins42 and found in all cases rather compact structures when Millikan’s 1.36 factor is included in the conversion between ion mobility and cross-sectional data; however, none as anomalously compact as the GroEL 14-mer (vide infra). This earlier work was less conclusive than the present study because the ions carried a substantial extraneous mass of adducts, with variations in mobility as high as 20% between the maximum in the peak and its high mobility end. An approximate idea of how much compaction has been achieved by the GroEL ions may be obtained by considering the extreme limit where the full protein mass mi ∼ 800.5 kDa has been compacted into a dense sphere. The density of such a sphere would be 1.17 g/cm3. Analysis by DMA-MS42 of smaller proteins and protein complexes (up to 150 kDa) shows that most have a gas-phase density similar to 0.95 g cm-3. The high density of GroEL ions is thus surprising, particularly in view of substrate protein binding data showing that the internal cavity of GroEL 14-mers is preserved in the gas phase.3 The recent study of Bush et al.,56 which involves many proteins and protein complexes, yields an approximate gas-phase protein ion density 0.86 g cm-3 (ion masses in the 12-336 kDa range, measured in N2 based on ξ = 1.36), but a GroEL ion density of 0.93 g cm-3. From the compilation of these two data sets, gathered with mobilities measured by two distinct instruments which differ in a number of ways, we confirm the conclusion by Bush et al.56 that GroEL is unique in terms of the compactness of its gas-phase structure. How such a large compaction may come about is presently unclear. While the large compaction factor determined for GroEL in these studies necessitates a focused computational analysis to fully assess the topology deformations possible for this system, we also note that our results are not inconsistent with previous results indicating that the binding cavity of the complex is retained in the absence of solvent. However, satisfying both our compact global measurements as well as the presence of a significant cavity places high constraints on the effective density of the protein complex. Specifically, in order to retain such a cavity within the complex, assuming a relatively isotropic compaction of the structure, the local densities of the subunits and interfaces that comprise the assembly will undoubtedly provide a
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physical limit to the compression experienced by the complex while retaining its barrel-like architecture. Further results and computational analysis are necessary to fully realize the potential gas-phase structures of the GroEL 14-mer suggested by the measurements described in this report. Comparison with Cross-Section Estimates Derived from Atomic Data. As a final validation of our conclusions regarding the compaction of GroEL 14-mer ion structure in the absence of bulk solvent, we estimated the collision cross section of the GroEL tetradecamer using atomic coordinates derived from X-ray diffraction data using a modified version of MOBCAL.2,26 The calculation provides two estimates of the orientationally averaged collision cross section, ΩPA and ΩEHSS, where the EHSS calculation estimates the effective drag by computing the influence of multiple specular collisions with neutrals during the ion mobility measurement and ΩPA estimates the cross section as a simple projection averaged over all orientations. ΩPA and ΩEHSS computed for the GroEL tetradecamer are 217.61 and 296.73 nm2, respectively. These calculations are in He gas, and though cross sections in air should be larger, the difference is modest given the large diameter of GroEL relative to those of either He or air molecules. Published calculations by an alternative method (using Visual Molecular Dynamics) give quite similar values (ΩPA = 220 nm2; ΩVMD = 300 nm2).3 The ratio of the two values (ΩEHSS/ΩPA) can be used to estimate the effective drag correction factor produced by the EHSS calculation for the GroEL X-ray structure, and compared to Millikan’s ξ = 1.36. The value obtained in the present EHSS calculation and that of ref 3 both give 1.36, in complete agreement with Millikan’s correction factor. We note, however, that this agreement applies to the crystal structure of GroEL rather than the unknown gasphase structure. Nonetheless, these calculations provide further evidence of the potential broad applicability of Millikan’s ξ to large proteins of various shapes.
’ CONCLUSIONS GroEL 14-mers electrosprayed under nondenaturing conditions from an aqueous ammonium acetate buffer were investigated by tandem differential mobility analysis-mass spectrometry (DMA-MS), leading to the following findings: 1. Very sharp mobility distributions may be achieved for rather large complexes such as GroEL without any declustering via proper sample purification. 2. It is therefore possible to obtain precise mobility data free from ambiguities usually associated with structural changes perhaps taking place at the entrance to the mass spectrometer. This point is highlighted by the presence in the DMA spectrum of two conformers of GroEL, while only one was previously found.3,56 3. The cross-sectional area of native GroEL is determined through multiple methods of calculation that converge to similar values, including the projection approximation and the geometric approach used here that exploits the symmetry and approximate geometric simplicity of this ion. 4. Our cross-sectional estimates of the X-ray structure and our cross-sectional measurements of the gas phase GroEL differ by more than 40%, indicating that electrospray ionization into dry air can lead to substantial compaction of the native structure. This conclusion is in contrast with prior work,3 even though the mobilities found in the two studies differ by much less than 40% (only 11% different). Most of the 3619
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The Journal of Physical Chemistry B disagreement stems from the inclusion in this work of the drag enhancement factor ξ, for which similar values are derived, either from Millikan’s work or from model calculations that consider multiple scattering events. 5. In spite of this quantitative disagreement, our findings are still compatible with prior observations that demonstrate the preservation of GroEL’s binding cavity, and thus the native topology and architecture of the complex, in the absence of bulk solvent.3 6. The collision cross section of the GroEL complex is found here to increase slightly with charge state, in a fashion that depends on the conformer and appears to be due partly to polarization effects, and perhaps also to Coulombic stresses.
’ ASSOCIATED CONTENT
bS
Supporting Information. Discussion on the effect of polarization/Coulombic stretching on the mobilities of GroEL 14-mers. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel: 203-432-4347. Fax: 203-432-7654. E-mail: juan.delamora@ yale.edu. )
Present Addresses
Currently at the Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN.
’ ACKNOWLEDGMENT We thank Alejandro Casado of SEADM and Bruce Thomson of MDS Sciex for the advice during the setup of the DMA-MS system and Joanna Freeke (University of Cambridge) for aiding in the MOBCAL calculations described. We are grateful to Dr. D. Goshtik of Applied Biosystems and G. Fernandez de la Mora of SEADM for the loan of the MS and the DMA, respectively, and to the Yale Keck Biotechnology Center for hosting the tandem instrument. B.T.R. acknowledges funding from Waters Corp. in the form of a research fellowship. C.V.R. is supported by a Royal Society Professorship. ’ REFERENCES (1) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658. (2) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S. J.; Robinson, C. V. Nature Protocols 2008, 3, 1139. (3) van Duijn, E.; Barendregt, A.; Synowsky, S.; Versluis, C.; Heck, A. J. R. J. Am. Chem. Soc. 2009, 131, 1452. (4) Pukala, T. L.; Ruotolo, B. T.; Zhou, M.; Politis, A.; Stefanescu, R.; Leary, J. A.; Robinson, C. V. Structure 2009, 17, 1235. (5) Uetrecht, C.; Versluis, C.; Watts, N. R.; Wingfield, P. T.; Steven, A. C.; Heck, A. J. R. Angew. Chem., Int. Ed. 2008, 47, 6247. (6) Fernandez de la Mora, J. Anal. Chim. Acta 2000, 406, 93. (7) Hogan, C. J.; Carroll, J. A.; Rohrs, H. W.; Biswas, P.; Gross, M. L. J. Am. Chem. Soc. 2008, 130, 6926. (8) Hogan, C. J.; Carroll, J. A.; Rohrs, H. W.; Biswas, P.; Gross, M. L. Anal. Chem. 2009, 81, 369. (9) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64.
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