Field Asymmetric Waveform Ion Mobility Spectrometry Studies of

21966

J. Phys. Chem. B 2006, 110, 21966-21980

Field Asymmetric Waveform Ion Mobility Spectrometry Studies of Proteins: Dipole Alignment in Ion Mobility Spectrometry? Alexandre A. Shvartsburg,*,† Tadeusz Bryskiewicz,‡ Randy W. Purves,‡ Keqi Tang,† Roger Guevremont,‡ and Richard D. Smith† Biological Sciences DiVision, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, and Thermo Electron Corporation, 1200 Montreal Road, Ottawa, Ontario K1A 0R6, Canada ReceiVed: April 26, 2006; In Final Form: August 16, 2006

Approaches to separation and characterization of ions based on their mobilities in gases date back to the 1960s. Conventional ion mobility spectrometry (IMS) measures the absolute mobility, and field asymmetric waveform IMS (FAIMS) exploits the difference between mobilities at high and low electric fields. However, in all previous IMS and FAIMS experiments ions experienced an essentially free rotation; thus the separation was based on the orientationally aVeraged cross-sections Ωavg between ions and buffer gas molecules. Virtually all large ions are permanent electric dipoles that will be oriented by a sufficiently strong electric field. Under typical FAIMS conditions this will occur for dipole moments >400 D, found for many macroions including most proteins above ∼30 kDa. Mobilities of aligned dipoles depend on directional cross-sections Ωdir (rather than Ωavg), which should have a major effect on FAIMS separation parameters. Here we report the FAIMS behavior of electrospray-ionization-generated ions for 10 proteins up to ∼70 kDa. Those above 29 kDa exhibit a strong increase of mobility at high field, which is consistent with predicted ion dipole alignment. This effect expands the useful FAIMS separation power by an order of magnitude, allowing separation of up to ∼102 distinct protein conformers and potentially revealing information about Ωdir and ion dipole moment that is of utility for structural characterization. Possible approaches to extending dipole alignment to smaller ions are discussed.

I. Introduction An electric field seeks to propel ions along the field direction. In a vacuum, the motion depends only on the ratio of ion mass (m) and charge (z); hence mass spectrometry (MS) methods1 of any technical implementation measure m/z. In a gas of sufficient number density (N), the dynamics is governed by ion mobility2 (K) that depends on the energy surface of interaction between an ion and a buffer gas molecule and the gas temperature, T. That surface is unique to each ion; thus the drift of ions in gases caused by electric field may be used to separate ionic mixtures, identify individual ions, and characterize the structures of unknown species. This fact underlies two analytical techniques, ion mobility spectrometry3-11 (IMS) and a more recent method of field asymmetric waveform IMS (FAIMS).12-59 In IMS, ions are separated by K while pulled through a known distance in a nonreactive buffer gas by a (typically) uniform electric field of a certain intensity (E).5 This is normally implemented in a drift tube, with the field created by a ring electrode stack along the axis. The value of K (termed K0 when reduced to standard conditions) is obtained from the steady-state drift velocity (V)

K0 )

P 273.15 V 760 T E

(1)

where P is the gas pressure. The mobility depends2 on E, which * Author to whom correspondence should be addressed. E-mail: [email protected]. † Pacific Northwest National Laboratory. ‡ Thermo Electron Corporation.

is important for dynamics at high E. In general, K(E) is an exceedingly complex function of collision integrals (Ω) of various orders formulated in the gas-phase transport theory.2 However, IMS is usually operated in the low-E limit where E is low enough for K(E) = K(0) and2

K0 )

( )

3ze 2π 16N µkBT Ω(1,1)

1/2

(2)

where e is the elementary charge, µ is the reduced mass of an ion/buffer gas molecule pair, kB is the Boltzmann constant, and Ω(1,1) is the first-order collision integral (cross-section). In FAIMS, ions are distinguished by the difference between K at high and low E. This is achieved by pulling ions (by gas flow or weak longitudinal electric field) through a space between the two electrodes (the analytical gap) carrying a periodic asymmetric waveform VD(t) that creates a field ED(t) in the gap.12-14 While the integral of ED(t) over the period is null, the mean positive and negative values of E differ. As the mobilities of ions depend on E, their displacements during the positive and negative ED(t) segments do not cancel exactly, and ions drift toward one of the electrodes and are neutralized on impact. For a particular species, the drift may be offset and the ions may be kept in equilibrium in the gap by superposing a small “compensation field”12-14 EC onto ED(t) by the addition of “compensation voltage” (CV) to VD(t). Scanning EC provides the spectrum of ions present, analogous to the operation of a quadrupole mass filter. The fundamentally optimum ED(t) profile is rectangular,17 but most FAIMS systems, including the

10.1021/jp062573p CCC: $33.50 © 2006 American Chemical Society Published on Web 10/06/2006

FAIMS Studies of Proteins

J. Phys. Chem. B, Vol. 110, No. 43, 2006 21967

Ionalytics Selectra used here, employ a more practical bisinusoidal waveform

π f sin(2πw t) + sin(4πw t - )]V [ 2 V (t) ) c

c

max

f+1

D

(3)

where wc is the frequency, Vmax (“dispersion voltage”) is the peak amplitude, and f controls the waveform profile. Most FAIMS systems (including Selectra and Sionex Differential Mobility Spectrometer) have adopted the optimum17 f ) 2. The EC for an ion depends on the carrier gas. Most work to date employed N2 (or air) or a He/N2 mixture;34,39,55 O2, CO2, N2/ CO2, He/SF6, and other compositions were also explored.25,28,30,38,53 In many gas mixtures (e.g., N2/CO2 or He/ SF6), high-field mobilities exhibit prominent non-Blanc phenomena that often materially improve FAIMS resolution.30,53 The K(E) function in any gas may be expressed as an infinite series

(

K(E) ) K(0) 1 + a

(NE) + b(NE) + c(NE) + ‚‚‚) 2

4

6

(4)

but terms beyond b(E/N)4 are generally insignificant2 at practical E. Depending on the signs of a and b, K(E) may increase (termed ions of type A) or decrease (type C); ions where K(E) has a maximum are called type B.13 Both cations and anions may be of any type.13,14 FAIMS analytical gaps have included planar,16,17 cylindrical,13,14 spherical,15 and a combination of cylindrical and hemispherical geometries.12 The electric field in a curved gap is inhomogeneous, focusing ions to the gap median.13,14,16,17 However, ED(t) of opposite polarities (called P1 and P2 modes for cations and N1 and N2 modes for anions) focus either A or C ions but defocus and eliminate the other from the gap.13,14 Ions of type B are normally focused as C, though EC has the same sign as for A. Thus Vmax and EC have opposite signs for types A (analyzed in the P1 and N1 modes) and B (in P2 and N2) but the same signs for type C (in P2 and N2). The above classification of ions is not fundamental but describes K(E) over the range of E sampled in FAIMS (j100 Townsend (Td)): eventually K(E) always decreases25 at some E. The type depends on the carrier gas; e.g., the heptadecanoic acid anion (269 Da) and tetrahexylammonium cation (355 Da) are A in N2O or CO2 but C in O2, N2, or SF6; Cs+ (133 Da) is A in N2, O2, N2O, CO2, or SF6 but C in He.25 Though FAIMS effectively separates isomers or isobars30-32 and is substantially orthogonal to MS in general,29,42 EC and m are statistically correlated: in any gas, ions shift toward type C with increasing25 m. The EC values of all ions previously studied by FAIMS (in N2 or air where significant statistics exists) are compiled in Figure 1. To the first order, EC scales59 as the cube of Emax, the value of E at the gap median when VD(t) ) Vmax. Hence, to compensate for the experimental variation of Vmax, we have plotted EC/Emax3, which reveals intrinsic FAIMS separation properties. (The EC also depends on the VD(t) profile, but all studies to date employed the waveform of eq 3 or a nearequivalent clipped-sinusoidal one.17) The quantity EC/Emax3 is negative for types A and B and positive for type C: ions shift to type C with increasing mass (Figure 1). Cations and anions exhibit close trends, and (in N2 or air) the transition from A to C occurs over the ∼100-350 Da range. B-type ions are found in that transition region, e.g., H+lysine33 (147 Da) and ketone derivatives20 such as H+(decanone) (157 Da) and H+(butanone)2 (145 Da). The data for He/N2 mixtures are scarcer than those

Figure 1. Dependence of FAIMS separation parameters in N2 or air on the ion mass for cations (filled circles) and anions (empty circles). The data include for cations Cs+,25 NO(H2O)+, NO2+, H+(H2O)3, H+N2(H2O)2, ethylamine, diethylamine, pyridine, and a silicon-based oil,13 triethyl-, tripropyl-, and tributylamines and their fragments,18 dimethyl methyl phosphonate and six aniline derivatives,45 methanol dimer, sodiated methanol and acetic acid,24 tetrahexylammonium,25 eight ketones from acetone to decanone and their dimers,20 eight aldehydes from propanal to nonanal,19 benzene, toluene, and ethyl- and propylbenzene,46 three xylene and two lutidine isomers,47 nine nitro-organic explosives,22 10 organophosphorus compounds and seven of their dimers,21 zarin,44 five piperidine derivatives,48 cisplatin and two of its complexes,38 six ephedra alkaloids,39 morphine and codein,34 cocaine,44 78 protonated and metalated disaccharides,40 19 amino acids,33 five polyglycines24 from G2 to G6, leucine enkephalin,24 bradykinin isomers,49,51 38 tryptic peptides (from BSA digest),29 four microcystins (cyclic heptapeptides),26 gramicidin S,35 and 39 bovine ubiquitin species32,57 (z ) 6-14) and for anions Cl-,31 CO3-, HCO3-, NO3-, and their heterodimers,13 three halogenates,23 (CH3O)CO2-, HSO4-, and acetate,24 ClO4- and H2PO3-,27 eight haloacetic acids,28 three phthalic acid isomers,30 nine naphthenic acids,50 five nitrobenzene explosives,43 19 amino acids,33 58 disaccharides and their adducts,40 and substance P (an undecapeptide) and its acetate adduct.24

for N2, but the qualitative trend is the same. In particular, all peptide ions have m J 350 Da and, regardless of z, are type C (in any gas tried).24,29,35,42,49,51 Less is known about FAIMS separations of protein ions. Previous work was limited to small (m e 16 kDa) proteins: ubiquitin,32,54-57 β2-microglobulin,52 hemoglobin chain A,12 and cytochrome c.12,57,58 For each, several conformers for almost every z generated by electrospray ionization (ESI) were resolved, all belonging to type C. In general, FAIMS is more orthogonal to MS than IMS and can distinguish multiple protein ion conformers where IMS finds only one (e.g., for ubiquitin 12+ and cytochrome c 13+ to 18+).55,57-60 FAIMS spectra of proteins are sensitive to homologous sequence substitutions12 and variations in solution conditions12,32 (even as small as increasing the organic solvent fraction32 from 45% to 50%), indicating that FAIMS can access the three-dimensional (3-D) structure and dynamics of proteins in solution. However, no calculation of high-field K(E) for polyatomic ions has been shown, which prevents the use of FAIMS for structural elucidation similarly to IMS.61-67 This problem is bypassed by the advent of two-dimensional (2-D) FAIMS/IMS analyses,42,57 which makes FAIMS separations of protein conformers much more topical. Here we present a broad investigation of FAIMS properties of protein ions, studying 10 common proteins in the 8-66 kDa range. We find a novel abrupt onset of pronounced B-type behavior for proteins above ∼30 kDa. We rationalize this as a manifestation of the (reversible) alignment of ions due to orientation of strong dipoles typical for large proteins by high electric fields in FAIMS, with the onset at ∼30 kDa consistent with a simple first-principles treatment.

21968 J. Phys. Chem. B, Vol. 110, No. 43, 2006

Shvartsburg et al.

II. Physical Foundation Mobilities of Ions Aligned by Electric Field. The crosssection Ω(1,1) that essentially sets IMS separation properties by eqs 1 and 2 is the orientationally averaged quantity Ω(1,1) avg : the average of directional cross-sections Ω(1,1) over all ion oriendir tations63 defined (assuming an atomic buffer gas) by three coordinate angles, φ, γ, and θ. However, θ is immaterial because the rotation around the E-axis does not affect Ω. The Ω(1,1) dir is an integral of a function of the scattering angle χ over the impact parameter b and the thermal distribution of relative velocities g between the ion and buffer gas molecule63

( )∫ ( )

π µ 4 kBT

Ω(1,1) dir )

3

∞

0

g5

-µg2 exp dg 2kBT

∫0

∞

[1 - cos χ(φ,γ,g,b)]b db (5)

Evaluating χ(φ, γ, g, b) requires modeling collision events for representative ion-molecule geometries. This has used methods of varied sophistication, ranging from (i) simple projection approximation (PA) computing the shadow area,62 to (ii) the EHSS model64 with hard-spheres interactions between atoms in the ion and in the buffer gas, to (iii) classical molecular dynamics in an approximated ion-molecule potential,63,65 and to (iv) scattering on electron density isosurfaces (SEDI) that represents an ion by a surface defined on a grid.66,67 In any case, the derivation of Ω(1,1) avg has invariably assumed equal probability of all ion orientations63

Ω(1,1) avg )

1 4π

∫0π dφ sin φ ∫02π dγ Ω(1,1) dir (φ,γ)

(6)

Virtually all large ions are not centrally symmetric and thus are permanent dipoles (p) that an electric field seeks to orient along E. However, thermal ions in gases rotate randomly. Hence, aligning the dipole depends on the work A needed to flip the dipole relative to the rotational energy, UR, available for that. For a dipole of moment p set at an angle a to E

A)

∫0π pE sin a da ) 2pE

(7)

There are two rotational axes orthogonal to p, and the mean thermal energy in each degree of freedom is kBT/2; thus 〈UR〉 ) kBT. The free rotation regime requires A , 〈UR〉 or

E , Ecrit )

0.5kBT p

(8)

In the opposite limit of E . Ecrit, the dipole is firmly locked. Between those extremes, ions exhibit a hindered rotation or pendular motion. When the inequality in eq 8 is not satisfied, we must modify eq 6 to weigh the probabilities of different ion orientations Pr(a). The angular coordinates for orientational averaging may be defined arbitrarily, and it is convenient to set φ ) a. Then

Ω(1,1) wei )

1 2π

∫0π dφ ∫02π dγ Ω(1,1) dir (φ,γ) Pr(φ)

(9)

where the (normalized) Pr (φ) is given by68

Pr(φ) )

Pr′(φ)

∫0

π

Pr′(φ)

Pr′(φ) ) exp

(

)

pE cos φ sin φ dφ (10) kBT

TABLE 1: Dipole Moments p for 10 Common Proteins: pnat Values Calculated72 for the Native Conformations from the PDB Obtained by X-ray and (Boldface) Solution NMRa calculations protein ubiquitin β2-microglobulin cytochrome cb cytochrome c (oxidized) cytochrome c (reduced) egg lysozyme hemoglobin chain Ab myoglobin milk β-casein carbonic anhydrase II liver alcohol dehydrogenase serum albumin

PDB code 1D3Z 1BD2 2B4Z 1AKK 1GIW 1E8L 1FSX 1G0B 1MYF 1CA2 3BTO: chain A chain B 1AO6: chain A chain B

measured nres 76 99 104 104 104 129 141 141 153 209 256 374 374 578 578

pnat, D 189 267 283 253 257 218 185 193 225

organism

bovine human bovine equine equine hen bovine equine sperm whale bovine 318 bovine equine 721 729 bovine 1139 1102

m, Da 8566 11 860 12 229 12 361 14 305 15 053 15 115 16 950 23 978 29 020 39 896 66 430

a Masses are from present measurements (the accuracy is approximately (1 Da, except approximately (10 Da for BSA). No geometries for bovine ubiquitin, carbonic anhydrase II, and serum albumin were found in the PDB; calculated p values are for the human homologues. Casein geometries for any organism were not found in PDB. b FAIMS spectra were also measured for the 18+ ions of canine, murine, and ovine cytochrome c (m ) 12 132-12 229 Da) and canine, murine, porcine, and human hemoglobin chain A (m ) 15 039-15 217 Da) (none found in the PDB).

In the limits of low p, low E, or high T where ions rotate freely, Pr′(φ) ) sin φ dφ, and eq 9 reduces to eq 6. Of course, for a (1,1) near-spherical ion Ω(1,1) wei would still be close to Ωavg , and large deviations from eq 6 will occur for substantially nonspherical species with p sufficient for alignment (pcrit). Coefficients a, b, ... in eq 4 that set K(E) at high E and thus control FAIMS separations are determined2 by combinations of higher-order collision integrals such as Ω(1,2), Ω(2,1), and Ω(2,2). Their evaluation has also involved full orientational averaging2 using eq 6. For aligned ions, those integrals become directional as well, and their calculation also calls for weighted averaging using eqs 9 and 10. Protein Dipoles. The dipole of a species is the sum of contributions from all polar bonds and net charges; thus statistically p increases for larger molecules. While typical p values for small molecules are 3.7 D (Figure 2). However, (i) proteins are never 100% helical, and (ii) helical elements are never fully aligned even in unfolded geometries.73,74 In fact, the unwinding of helices during unfolding randomizes the dihedral angles,74 degrading the alignment of peptide bond dipoles, and thus should reduce p for substantially helical proteins. Dipole Alignment of Protein Ions in FAIMS. In present IMS systems, E equals 2-30 V/cm for “low-pressure” IMS/ MS operated at j10 Torr7-9,11,42,60,62,63,65 and ∼50-700 V/cm for “high-pressure” IMS and IMS/MS at ∼150-760 Torr.4,9,75-77 By the condition in eq 8, at room temperature ions with p , ∼8000 D exhibit free rotation at any E < 700 V/cm. Hence no dipole alignment is expected in IMS, except possibly for extraordinarily large ions and charged particles. Indeed, all IMS measurements to date are consistent with orientational averaging by eq 6. In contrast, typical Emax values for FAIMS are 16-25

Figure 3. Scheme of the dipole locking progressing from a to d as p and/or Emax increase.

kV/cm,12-59 which should substantially align ions with p J 250-400 D (Figure 3a). To become relevant, such an alignment has to occur over a significant part of ED(t). For example, a 20% portion means Ecrit ) 0.642Emax (with the waveform of eq 3 and f ) 2), leading to a pcrit ≈ 390-620 D (Figure 3b). Hence, most protein ions will experience substantial dipole alignment under standard FAIMS conditions. In Table 1, those include alcohol dehydrogenase, ADH (pnat >720 D), bovine serum albumin, BSA (pnat >1100 D), and likely also carbonic anhydrase II, CA II (pnat ) 318 D, appears short of the predicted pcrit, but a moderate unfolding may raise p above that threshold). Statistically (Figure 2), dipoles should align for (native) proteins with nres J 170-300 (or m J 20-33 kDa). However, there is a substantial scatter (r2 ) 0.58) and outliers; many proteins have m < 20 kDa but p > 600 D or m > 33 kDa but p < 400 D (Figure 2). For example, human GABARAP protein (1KLV) has nres ) 117, m ) 13.9 kDa, and p ) 815 D, while duck apo-ovotransferrin (1AOV) has nres ) 686 and m ) 75.8 kDa but p ) 359 D. The effect of dipole alignment on FAIMS analyses depends on whether E varies slowly enough for the ion orientation to adjust. When E changes sign (or |E| drops below the threshold for dipole locking), flipping the dipole orientation (or randomizing it by thermal rotation) takes time. This relaxation time (trel) should be compared with the ED(t) period, tper ) 1/wc. If trel , tper, then the dipole alignment is governed by instantaneous E. If trel . tper, then the alignment is set by mean ED(t) ) 0, and dipoles are not oriented (as in the free rotation regime). In intermediate cases, the alignment behavior becomes hysteretic, following ED(t) with a time lag, with the mean extent of alignment lower than that when trel , tper. With typical wc )


Shvartsburg et al.

200-750 kHz in FAIMS,12-59 tper ≈ 1.3-5 µs. The trel time has two components: tin due to inherent inertia of an ion (regardless of the media) and tvis due to viscous gas friction (depends on the gas identity and pressure). The magnitude of tin may be estimated as one rotational period. For a uniform sphere of radius rsp, the moments of inertia are

I)

2mrsp2 5

(11)

and mean frequencies of thermal rotation (around each of three axes) are

wr )

( ) kBT I

1/2

(12)

then

tin )

2π ) 2πrsp[2m/(5kBT)]1/2 wr

(13)

For the protein geometries in Table 1, we found rsp ≈ 18-33 Å, and, by eq 13, tin ≈ 0.4-2.2 ns. The value of tvis is calculable from the ion volume (V) and gas shear viscosity, η0 (1.74 × 10-5 Pa s for air or N2 at standard temperature and pressure), using the Stokes-Einstein-Debye equation78,79

η0V kBT

(14)

4πη0rsp3 3kBT

(15)

tvis ) For a sphere of radius rsp

tvis )

producing (for rsp ≈ 18-33 Å) tvis ≈ 0.1-0.6 ns and tvis < tin at any rsp. Hence the relaxation dynamics is mostly controlled by inertia with little dependence on the FAIMS gas, and trel < tin + tvis ≈ 0.5-3 ns for the near-native conformers of proteins in Table 1. These values are ∼1/1000 of the shortest tper ) 1.3 µs, allowing essentially no hysteretic behavior. Unfolding of a protein will increase both tin (because of higher IR along at least one axis) and tvis. To gauge the maximum principal I for extended proteins, we assume linear “ribbons” with the length of L ) 3 Å × nres, which is longer than unfolded geometries produced by molecular dynamics at the highest experimental z (for cytochrome c).74 Then

mL2 Imax ) 12

(16)

and

tin ) πL

[ ] m 3kBT

1/2

(17)

For proteins in Table 1, eq 17 translates into tin ≈ 2.4-51 ns. The times at the upper end of that range are much greater than those for native conformers because of very high Imax values for straight chains with hundreds of residues spanning ∼103 Å, a rather extreme scenario. Still, trel < 0.03tper, meaning marginal (if any) hysteretic effects.

Even for extremely large proteins, such effects will be minor for reasonably compact geometries. For example, a hypothetical spherical protein with m ) 1 MDa would have rsp ≈ 77 Å, producing tin ≈ 20 ns by eq 13 and tvis ≈ 8 ns by eq 15, with the resulting trel still ∼2% of the shortest tper. An extended peptide strand of that mass would be ∼27 300 Å in length (assuming 110 Da/residue), and by eq 17, tin ≈ 3.1 µs. That value is comparable to typical tper values in FAIMS, meaning a strong hysteresis that drastically affects the ion alignment and largely divorces it from the instantaneous E. However, real proteins of this size are multistranded,80 making this situation unlikely in practice. Thus, although caution is advised for very large and/or unfolded proteins, to the first approximation the orientation of most protein ions in FAIMS may be deemed controlled by current E only, and that is assumed in the following discussion. With increasing p and/or Emax values, initially the dipole will lock at the ED(t) peak but freely rotate elsewhere (Figure 3a). Then the region over which locking takes place will broaden (Figure 3b), and eventually the dipole will also lock (in the opposite direction) at maximum |E| in the low-E half-cycle (Figure 3c). This occurrence depends on the ED(t) profile that sets the ratio of Emax to that maximum |E|. With eq 3 and f ) 2, the p needed for locking in the low-E half-cycle equals 2pcrit, e.g., ∼500-800 vs ∼250-400 D. With f * 2, the required p would be different, e.g., ∼420-670 D with f ) 4 in some experiments29 (where the maximum |E| in low-E half-cycle equals 0.6Emax). With any f, a material effect will again require alignment over a significant portion of the half-cycle and thus higher p. As p and/or E increase further, locking would occur over yet larger portions of both FAIMS half-cycles, with free rotation remaining only near the points of orientation flipping where E changes sign (Figure 3d). Of course, dipole locking is not a quantum jump but rather a gradual transition from free rotation to hindered rotation to pendular oscillations with progressively smaller amplitudes, and the above discussion is merely to illustrate possible regimes for the dipole alignment in FAIMS. In principle, ions in an electric field develop an induced dipole pin that depends on polarizability. In general, pin for an oriented ion is set by the polarizability tensor and is at an angle to p, and the total dipole pt (the sum of p and pin) is not exactly parallel to p. Since E actually seeks to align pt rather than p, formally the orientation of ions in FAIMS is controlled not just by the permanent dipole but also by the polarization properties. However, magnitudes of pin for proteins are negligible compared to p, even at peak E in FAIMS. Indeed, the dielectric constant for bulk proteins is81 ∼2-4, and the susceptibility χe equals ( - 1) ≈ 1-3. Using81

pin ) 0χeEV

(18)

we find pin ranging from 0.2 D for ubiquitin to 1.4 D for BSA (even at E ) Emax and χe ) 3). Those values are ∼0.1% of p for these proteins (Table 1), and the applicability of eq 18 to single protein molecules has been validated by sophisticated calculations.81 Hence, the alignment of protein ions in FAIMS is essentially governed by the permanent dipole only. More importantly, the force of E propelling an ion acts on the center of its charges, while the drag force is applied to the center of mass. In general, those two centers do not coincide, which creates a torque seeking to orient the vector connecting them along E. That torque could be accounted for by using p with respect to the center of mass, which differs from p computed by many utilities including the server to calculate



Figure 4. Views of HSA chain A from three orthogonal directions (x (1,1) is set along the dipole p). Values of Ω(1,1) dir for each view and Ωavg (in Å2) are calculated using PA and EHSS (underlined), assuming the collision radii of 3.2 Å for H and 3.7 Å for other ion atoms, as appropriate for N2 buffer gas. The orientations of p are shown for each panel.

protein dipole moments.72 However, the difference is usually modest and not critical to this work. Another potential hysteresis16,82 has to do with the time (τ) needed for the drift velocity to adjust to changing E. When E varies so fast that the drift conditions are not steady-state, the mobility depends not just on instant E (by eq 4) but also on past E, which would narrow the sampled range of K and thus adversely impact FAIMS performance. The value of τ is16,82

τ)

2mK(E) z

(19)

which remains valid for oriented dipoles. For large molecules, Ω(1,1) scales with dimensions squared and thus with m2/3, while z values for protein ions generated by ESI are roughly proportional83 to m1/2. Then τ is proportional to m-1/6; i.e., the phase shift between ED(t) and K(t) diminishes for larger protein ions. For species considered here, τ ≈ 2-4 ns for compact geometries and yet less for unfolded ones. This is under 0.3% of the shortest tper in FAIMS, meaning negligible hysteretic effects. Effect of Dipole Alignment on FAIMS Separation Parameters. The alignment will always be stronger at higher 〈E〉, i.e., in the high-E half-cycle. The impact on EC depends on whether p and the “long” molecular rotation axis are closer to (i) collinear or (ii) orthogonal: ions are oriented “edgewise” (1,1) (Ω(1,1) < Ω(1,1) > p avg ) increasing K in i and “broadside” (Ωp (1,1) (1,1) (1,1) Ωavg ) decreasing K in ii, where Ωp is Ωdir in the plane orthogonal to p. The effect is greater at higher E in either case; hence there will be a shift toward type A or B in i and toward type C in ii. Class i is illustrated by the native conformation of human serum albumin (HSA) chain A (Table 1 and Figure 4). As is usual for proteins, EHSS64 cross-sections exceed PA62 values by ∼20%. However, relative Ω values are consistent in both treatments, and Ω(1,1) (top left panel) is lower than Ω(1,1) p avg by ∼9-10% (Figure 4); i.e., native BSA belongs to class i.

Class ii may be exemplified by hen ovotransferrin (1OVT, nres ) 682, m ) 76.0 kDa) with p ) 825 D, where calculated Ω(1,1) p exceeds Ω(1,1) avg by 17-20%. Unfolded globular proteins commonly have little tertiary structure but retain substantial helicity74 and thus may have a large p sufficient for dipole alignment; the difference between Ω(1,1) and Ω(1,1) p avg for such geometries may be yet greater than those in the above examples. In reality, the alignment is never perfect (as shown in Figure 4) because of pendular oscillations, and Ω(1,1) wei produced by eq 9 will generally be closer to Ω(1,1) . avg At very high p, when dipoles become locked over most of the low-E half-cycle (Figure 3d), K in both half-cycles will only, and the effect of alignment increasingly depend on Ω(1,1) p on FAIMS separations will abate. In the limit of infinite p, FAIMS will measure the ratio of Ω(1,1) at high and low E, p which should be not too far from the same for Ω(1,1) avg of (1,1) unaligned ions (even when the absolute Ω(1,1) and Ω are not p avg close). The dipole alignment effects are superposed on K(E) for orientationally averaged ions. In ii, the shift toward C-type behavior due to alignment will augment the “normal” C-type behavior of large ions. Since the magnitude of EC for normal C-type behavior is not known a priori, convincingly demonstrating that shift experimentally would not be trivial. In i, the shift toward A-type behavior will compete with factors that normally render large ions C-type. However, the typical decrease of K for small proteins over the range of E relevant to FAIMS is ∼1-2%, i.e., an order of magnitude less than the possible increase of K caused by alignment. Hence, even a limited dipole alignment could raise K more than enough to offset its decrease at high E for freely rotating macroions, thus producing prominent A- or B-type FAIMS behavior for large proteins with sufficient dipole moments. Here we tested this hypothesis by measurements for the representative proteins (Table 1). III. Experimental Methods The experiments employed an ESI/FAIMS/MS instrument comprising a Selectra FAIMS system and the LCQ Deca XP Max ion trap mass spectrometer (Thermo Electron, San Jose, CA). The FAIMS and MS stages were coupled via a small chamber mounted on the capillary inlet to the mass spectrometer. The ESI emitter of the LCQ was pulled back and positioned a short distance away from the FAIMS entrance. The ESI was operated with a voltage of ∼5 kV and moderate sheath and auxiliary gas flows. The Selectra device was described previously.12,84 Briefly, the 2 mm analytical gap is defined by coaxial cylindrical electrodes with radii of 8 mm (internal) and 10 mm (external). The gap in the hemispherical section (adjusted by internal electrode translation) was ∼2.1 mm, which provides nearmaximum FAIMS sensitivity at the cost of limited resolution.84 Ions entered the analytical gap through a curtain gas interface. The N2 gas flowed in through a side opening in the interface and split into two streams: The major stream exited through the curtain plate orifice and desolvated incoming ions while the minor stream carried ions into the sampling orifice and through the gap. The unit supplying the gas dried and purified it and set the total flow rate to 2.5 L/min, with ∼0.5-0.6 L/min flowing through the gap. That flow was less than the ∼0.8 L/min conductance of the (0.5 mm i.d.) MS inlet capillary, and a makeup (N2) gas was added to the FAIMS/MS interface chamber. At these conditions, ions traversed the FAIMS instrument in ∼0.2-0.4 s. The VD(t) value by eq 3 (wc ) 750 kHz) and CV were coapplied to the inner FAIMS electrode. Separations were

21972 J. Phys. Chem. B, Vol. 110, No. 43, 2006 performed in the P2 mode using a Vmax value of -4 or -5 kV (|E| of 2.0 or 2.5 kV/mm), with a CV scan speed of 5 V/min. The whole FAIMS unit was biased at the MS inlet voltage, kept low (5-50 V) to minimize ion heating in the skimmer region and thus avoid any processes (e.g., proton stripping or protein oligomer dissociation) that could distort the results. The LCQ was operated in the MS mode. The inlet capillary was not heated (since ions are desolvated prior to FAIMS). The pressures in the skimmer and analyzer chambers, as measured by a convectron and ion gauges, were ∼1 Torr and ∼15 µTorr, respectively. The MS spectra for ions filtered by FAIMS were obtained by scanning the m/z ) 200-2000 range or its segments corresponding to specific z values (each acquisition comprising 10 scans with a maximum ion injection time of 0.1 s). FAIMS spectra for all z values (the total ion count, TIC) or particular z values were extracted using the LCQ software. The proteins in Table 1 (except microglobulin) were purchased from Sigma (St. Louis, MO). Powders were dissolved in water (w), water/methanol (w/m) mixture, or acetonitrile (An) to 1-30 µM and acidified by 0-5% acetic (Ac), formic, or hydrochloric acid, as specified. For most proteins, we analyzed at least two samples. In one, minimizing the disruption of native conformations, the solvent was mostly aqueous with no or minimum organic content and near-neutral pH (“pseudonative” media).85 In the other, maximizing protein denaturation, we used solvents with the highest organic fraction and the lowest pH (“denaturing” media),85 with some samples annealed at ∼80 °C for 1-2 h. Conditions between those extremes should generally result in intermediate conformers, as shown for CA II solutions with five different pH values. Some proteins studied have covalent disulfide (S-S) bonds that constrain the conformational freedom. In the “denatured” samples, those were reduced by addition of 0.1-5 mM dithiothreitol (DTT) and heating as described above. Solutions were infused into the ESI emitter by the LCQ syringe pump at 1 µL/min. IV. Results Below we describe the FAIMS data in the order of increasing protein mass (data for microglobulin are from ref [52]), showing TIC spectra that reveal the whole EC range across the envelope of z values generated by ESI and the spectra for each z value observed. The latter are compiled into 2-D FAIMS/MS maps for conciseness, with CV spectra for each z value in the Supporting Information. All FAIMS spectra are scaled to unit peak height, but the true intensities are apparent from the mass spectra. However, relative heights of the CV spectral peaks (especially at very different CVs) are not quantitative,16,23 because the ion focusing in cylindrical FAIMS depends on CV.16,17 Still, the trends of isomer abundances (e.g., as a function of solution conditions) that involve no CV change should be accurate. Conventional FAIMS Behavior for Small Proteins. Ubiquitin is a eukaryotic cytoplasmic protein containing an R-helix, a 3(10)-helix, and a five-stranded β-sheet. Ubiquitin lacks S-S bridges and so is flexible, exhibiting a rich conformational diversity. ESI from pseudonative and denaturing solutions generates ubiquitin ions with z ) 5-8 and 6-15, respectively.55,85 From IMS data,59 ion geometries are folded for 5+, mostly unfolded for z g 11, and range from folded to unfolded for z ) 6-10. Extensive FAIMS data for bovine ubiquitin ions in N2 (for z ) 5-15) and in He/N2 (for z ) 11-15) were reported.32,54-57 For all z values, FAIMS distinguished multiple isomers,32,55,57 all being C-type in N2, 1:1 He/N2, and 3:2 He/ N2 (EC ) -(2-10) V/mm at Emax ) -(1.65-2.2) kV/mm).

Shvartsburg et al.

Figure 5. FAIMS spectra for cytochrome c ions from 50 µM solution in (1:1 w/m + 1% Ac). Charge states are labeled.

Presently, we have studied ions with z ) 5-14 produced from pseudonative (1:1 w/m + 0.1% Ac) and denaturing (1:1 w/m + 1-5% Ac) media. The results are close to the published data, except |EC| is higher at (5-14) V/mm because of higher |Emax| ) 2.5 kV/mm. Human β2-microglobulin (m ≈ 12 kDa) with the β-sandwich structure and a single S-S link is fibrillogenic.52 For this protein, ESI produced 7+ and 8+ ions from neutral and z ) 7-14 from acidic solutions.52 While S-S bridge(s) prevent(s) a complete unfolding of a protein, partial unfolding may still occur in solution52 or in the gas phase.85 The resulting conformers could be characterized by IMS, e.g., as for lysozyme.85 Microglobulin denatures in solution at pH < 5, and FAIMS (in 1:1 He/N2) found52 at least two isomers for 8+ and 12+. The peaks at a lower |EC| values were tentatively assigned to the partially folded amyloidogenic conformer (for 8+) and acid-unfolded species (for 12+).52 Again, all isomers for all z values exhibited C-type behavior (EC ) -(8-14) V/mm at Emax ) -2 kV/mm). Overall |EC| did not decrease for higher z values significantly (unlike for ubiquitin), perhaps because the S-S bridge prohibited full protein unfolding. Cytochrome c is an electron-transfer metalloprotein (m ≈ 12 kDa) with no S-S bridges, comprising two R-helices with the rest wrapped around the heme. For bovine or equine cytochrome c, ESI generates z ≈ 6-19, and the unfolding occurs at z ≈ 9-11 (from IMS data at conditions most likely to preserve solution structures, a water solution and the lowest IMS injection energies).86 FAIMS spectra of cytochrome c 18+ from five mammals (Table 1) in N2 exhibit several conformers, all C-type (EC ) -(2-4.5) V/mm at Emax ) -2.15 kV/mm).58a Further, conformers of equine cytochrome c are C-type for all z values generated by ESI, based on EC ) -(1-7) V/mm measured at Emax ≈ -2 kV/mm for z ) 9, 11, 13, 16-18,58b z ) 10-15,57 and z ) 7-9 herein (Figure 5). Unfolding of cytochrome c tends to reduce |EC|, shifting it from 5 to 6 V/mm for compact geometries (z e 9) to 2-4 V/mm for extended ones (z g 9). This is similar to the data for ubiquitin, where |EC| (at same Emax ) -2 kV/mm) decreases from 5 to 8 V/mm for compact 6+ and 7+ ions to 3-5 V/mm for unfolded proteins with z g 11. Lysozyme is an enzyme (m ≈ 14 kDa) that ruptures bacterial cells. It contains five helices, five β-sheets, and a large amount of random coil and β-turns, with four S-S links.85 ESI of disulfide-intact (di) lysozyme yields z ) 8-11 with a compact near-native shape, as revealed by IMS when ions are injected gently to preserve the source conformers.85 Annealing of ions by collisional heating on energetic injection into IMS produces less compact (but still folded) conformers. Present FAIMS spectra for z ) 8-10 were obtained without annealing and show a single isomer (Figures 6c and 6e). For disulfide-reduced (dr) lysozyme,85 ESI yields z ) 10-18. The MS distribution is



Figure 6. Lysozyme MS (top panels), FAIMS (middle), and FAIMS/MS (bottom) spectra. Charge states are labeled. An inset in the MS panel shows the spectrum scaled as indicated. The left panels are for di-ions from 1 µM solution in (1:1 w/m + 2% Ac), and the right panels are for dr-ions from 30 µM in (1:1 w/m + 5% Ac + 1.5 mM DTT).

bimodal (Figure 6b), with a part due to di-protein centered at 9+, indicating that S-S links were severed incompletely or reformed before analysis. As for ubiquitin and cytochrome c, |EC| decreases from 11 to 8 to 6-7 V/mm as lysozyme unfolds with z increasing from 8-10, to 10-13, and to 13-20, respectively (Figure 6f). The two species for 10+ apparently are the di and dr forms. The origin of a smaller EC shift at z ) 13 (and the corresponding two 13+ isomers) is not clear: 13+ is not special in IMS data.85 All di- or dr-lysozyme ions are C-type. Hemoglobin is an Fe-containing oxygen-transport protein in blood erythrocytes. The normal adult hemoglobin comprises two A and two B chains, with eight or nine R-helices each. The 18+ ions of chain A (m ≈ 15 kDa) exhibit up to 10 conformers resolved by FAIMS (in 3:7 He/N2).12,58 Though >50 positions in chain A are conserved for all mammals, the spectra for five out of six species (Table 1) are quite distinct. Still, all chain A ions are C-type (EC ) -(1-8) V/mm at Emax ) -2.15 kV/ mm). Myoglobin, found in muscle tissues, is another Fe-containing oxygen transporter that has no S-S bridges and thus adopts preferred conformations. With m ≈ 17 kDa, it is the largest protein probed by either IMS/MS or FAIMS/MS previously. ESI of sperm whale myoglobin solutions (in 3:1 w/An + 0.25% Ac) produced z ) 8-22, with unfolding at z ) 7-10 (by IMS data).87 However, a harsh solvent and energetic IMS injection in those experiments promoted unfolding that otherwise would likely occur at higher z values. Here we used a 100% aqueous “pseudonative” media and a highly denaturing solution in hot An. As expected, denaturation i shifts the distribution of ions to higher z value, 9-23 with maximum at 13+ for An solution

(Figure 7b) vs 9-19 with maximum at 11+ for aqueous media (Figure 7a), and ii broadens FAIMS spectral features (with mean full width at half-maximum increasing from 1.7 V/mm in Figure 7e to 2.8 V/mm in Figure 7f), which indicates a greater conformer diversity. Otherwise, the two solutions give rise to similar FAIMS spectra. All myoglobin ions are C-type, and as for other proteins lacking S-S links studied above, |EC| gradually decreases with increasing z (from 6-8 to 5-6 V/mm over z ) 9-23). Caseins (m ≈ 19-25 kDa) are the predominant milk phosphoproteins. With no S-S links and little fixed 3-D structure, they are flexible and do not clearly denature but rather assume various geometries depending on conditions. As a result, caseins are among proteins that do not crystallize, and their native conformations are difficult to determine (thus no pnat value is given in Table 1). Bovine β-casein (m ≈ 24 kDa) is amphiphilic, and 35 out of 209 residues are prolines that do not interact, hence a particularly disordered structure. Despite those unusual properties, data for β-casein are not remarkable: ESI of an acidified solution yields z ) 13-22 with a maximum at 16+ (Figure 8a). All those ions are C-type in FAIMS, and following the above pattern for proteins with no S-S bonds, |EC| drops from ∼7 to ∼5 V/mm as z increases (Figures 8b and 8c). “Abnormal” FAIMS Behavior for Large Proteins. Carbonic anhydrases are Zn-containing enzymes catalyzing CO2 hydration and maintaining the pH balance that are particularly widespread in erythrocytes. A common CA II (m ≈ 29 kDa) is a 10-stranded antiparallel twisted β-sheet surrounded by seven R-helices. ESI of neutral aqueous CA II produces a distribution with z ≈ 15-35 and maximum at ∼22+ (Figure 9b). Adding


Shvartsburg et al.

Figure 7. Same as Figure 6 for myoglobin, 4 µM solution in (w + 0.1% Ac) (left panels) and 4 µM hot solution in (An + 0.1% HCOOH) (right panels).

just 0.01% Ac creates another distribution centered at z ≈ 3334 (Figure 9d); its intensity grows with increasing acidity up to ∼30% of the total at ∼0.5% Ac (Figure 9f) but remains constant at lower pH (Figure 9h). Appearance of such bimodal ESI/MS distributions upon acidification is common for proteins:75 unfolded conformers have more protonated sites and thus higher z values. However, FAIMS spectra for CA II (Figures 9a, 9c, 9e, and 9g) are without precedent for proteins or any large ions: besides the usual C-type feature at negative EC, there is a B-type peak at positive EC. Relative intensities of the new peak and MS envelope around z ≈ 34 are proportional at all pH values (Figures 9a-h), so B-type ions must be unfolded proteins with z J 30. That conclusion is confirmed by FAIMS/MS data (Figures 9i and 9j). From the neutral solution (Figure 9i), CA II ions with z j 30 behave like those for smaller proteins above, with EC shifting from -(8-9) to -(5-6) V/mm as z increases from 15 to 30. The B-type peak at EC ≈ 18-20 V/mm appears at z ≈ 28 and becomes dominant at the highest z observed (34+ and 35+). For z ) 30-35, the signal intensity at all EC values between the two peaks is substantial (Figures 9a and 9i), apparently due to trapped intermediates on the unfolding pathway(s). (Such “partly folded” geometries are ubiquitous in the IMS spectra of protein ions with z in the unfolding transition region.) Reducing the solution pH favors unfolded conformers and thus should lower the range of z over which they emerge in ESI/FAIMS. Indeed, with denaturing media, some fraction of “unfolded” and “partly folded” species appears at all z (Figure 9j), and the major transition to B-type conformers proceeds over z ≈ 25-32, with >85% ions for z ) 32-38 being B-type. At the highest z ) 39, that fraction apparently decreases to ∼70%, which may be an artifact of a low signal-to-noise ratio at the edge of the z distribution.

Figure 8. Same as Figure 6 for β-casein, 2 µM in (1:1 w/m + 1% Ac).



Figure 9. CA II FAIMS (a, c, e, and g) and MS (b, d, f, and h) spectra for 1 µM solutions in w (a and b), w + 0.01% Ac (c and d), w + 0.5% Ac (e and f), and w + 5% Ac (g and h). FAIMS spectra for panels b and f are shown in panels i and j, respectively. The white bar separates B-type conformers (to the left) from C-type (to the right).

The new B-type behavior is dominant for the two larger proteins that we studied: ADH and BSA. Found in many organisms, ADH facilitates the conversion between alcohols and aldehydes or ketones, including the oxidation of ethanol in

humanssthe rate-limiting step of alcoholic beverage metabolism. In mammals, ADH is contained in the stomach lining and liver. ADH is a dimer of (nearly) identical strands, each containing R-helices, β-sheets, and two Zn2+ ions (with one in


Shvartsburg et al.

Figure 10. Same as Figure 6 for ADH, 25 µM in w + 0.05% Ac (left panels) or in An + 0.1% HCOOH (right panels). The white bar and dashed line separate B-type conformers (to the left) from C-type conformers (to the right).

the active site). The EE form of horse liver ADH studied here is a homodimer of E strands with m ≈ 40 kDa each. By the MS spectra under all conditions tried (e.g., Figures 10a and 10b), the dimer dissociates during the ESI process. For slightly acidified aqueous ADH solutions, ESI produces an apparently bimodal distribution of z from 34 remain C-type (with EC ≈ -(7-9) V/mm) (Figure 10f). Unless some compact conformers improbably persist up to the highest z even when derived from an extremely denaturing 100% aprotic solution, some unfolded ADH geometries are C-type.

As we discussed, those may be species with mostly destroyed secondary structure and thus randomized peptide bond orientations, resulting in a low p and thus little dipole alignment, or ions with very high p that shifted to type C because of strong dipole locking over both FAIMS half-cycles. BSA is the prevalent blood plasma protein that mainly regulates the colloidal osmotic pressure and buffers blood pH. Native BSA (m ≈ 66 kDa) is heart-shaped and consists of three homologous domains, each comprising one subdomain with six R-helices and another with four. The overall helicity is ∼2/3, with the remainder made of turns and extended/flexible regions between subdomains but no β-sheets. BSA has 17 S-S links, mostly between helical segments. ESI of di-BSA produced z up to ∼50, but observation of z < 34 was precluded by instrumental limitations, and the distribution maximum could not be determined (Figure 11a). The FAIMS behavior of di-BSA resembles that of denatured ADH (Figure 10f). For all z ) 34-50, there are typical C-type ions at EC ) -(5-8) V/mm, prominent B-type conformers around ∼14-16 V/mm, and a broad range of species at intermediate EC (Figures 11c and 11e). The B-type fraction increases from ∼60% to ∼80% over the z ) 34-44 range, then remains at ∼80% for higher z values (Figure 11e). Severance of S-S links extends the charge distribution to z ≈ 60 (Figure 11b). For all ions with z ) 34-50, common with di-BSA, the B-type fraction for dr-BSA is 45-60% (increasing at higher z), i.e., always less than that for di-BSA. For z > 50, that fraction is ∼50-55%. The measured partitioning of CA II, ADH, and BSA ions between types B and C is summarized in Figure 12. (We caution that the data are for specific z values as shown but should not be extrapolated to the whole ion signal as the instrumental



Figure 11. Same as Figure 10 for BSA, 15 µM in 1:1 w/m + 0.25% Ac (left panels) or 25 µM in 1:1 w/m + 0.1% HCl + 5 mM DTT (right panels).

Figure 12. Percentage of ions that belong to B-type for CA II (circles), ADH (diamonds), and BSA (triangles), with the pseudonative media (filled symbols) and denaturing media (empty symbols).

limitations precluded measurements for low z values.) As reported above, denaturation in solution drastically increases the B-type fraction for CA II or ADH but decreases it for BSA ions (at any z). Elimination of S-S bonds should make proteins less compact. Hence (full or partial) unfolding apparently converts some BSA ions from B- to C-type conformers, the trend opposite to that for CA II and ADH. This difference may be due to a much higher pnat value for BSA compared to that for CA II or ADH (Table 1). That could produce an effective FAIMS alignment of most compact conformers generated from pseudonative media for BSA but not for CA II or ADH. As

Figure 13. Same as Figure 1 for peptides and proteins, including the data from Figure 1 and present measurements. As protein ions have a large number of conformers (across the charge state distribution) that densely cover a range of EC values, separation parameter ranges are plotted.

mentioned in the “protein ion dipoles” section, unfolding may either elevate p (e.g., by aligning antiparallel helices) or reduce p (e.g., by destroying helices). Statistically, this randomization process would populate isomers with p > pcrit when p < pcrit for the starting geometry (or their majority in the initial ensemble) and depopulate those otherwise. Then an extensive denaturation should, in general, increase the B-type fraction when it was originally small (as for CA II or ADH) but decrease it when it was large (as for BSA). Denaturation of large proteins with high pnat values such as di-BSA may also create abundant conformers with very high p value (that may shift to type C because of dipole locking in both FAIMS half-cycles). However, the EC value of such conformers would generally differ from

21978 J. Phys. Chem. B, Vol. 110, No. 43, 2006 that of unaligned ions, because Ωp and Ωavg would not depend on E in exactly the same way. Indeed, the dominant FAIMS peak for dr-BSA is at EC ≈ -2 V/mm (Figure 11f) versus approximately -6 V/mm for C-type ions of di-BSA (Figure 11e). Thus, more experiments and modeling are needed to understand the apparent depopulation of B-type conformers upon BSA denaturation. V. Conclusions Summary of Results. We have surveyed FAIMS separation properties of selected protein ions spanning the ∼10-70 kDa range. To cover the broadest range of solution structures, samples were prepared using both nativelike and highly denaturing media. Regardless of those conditions and the charge state z, all protein conformers with masses up to ∼27 kDa belong to FAIMS type C, where ion mobility K (in N2 and other gases tried) decreases over the range of electric field intensities E. Such behavior is well-established for ions heavier than ∼400 Da (in N2), including peptides and proteins. This typical behavior includes an overall decrease of absolute compensation field (EC) with increasing z. We have found that larger proteins display a strikingly different FAIMS behavior. The three proteins above ∼27 kDa (carbonic anhydrase II, alcohol dehydrogenase, and albumin) exhibit abundant B-type conformers. Those species have EC up to ∼25 V/mm (Figures 10f and 11f), an extremely high value comparable only to those for smallest ions with a few atoms. This new “abnormal” behavior contrasts with that of peptides and smaller proteins (Figure 13). We rationalize this behavior as a consequence of reversible locking of electric dipoles of protein ions by the FAIMS electric field. Most proteins have large dipole moments p, because of contributions of many ordered peptide bonds and physical dimensions that permit large distances between net charges. The protein size and p are statistically correlated, and for the native conformers, p typically ranges from ∼102 D for small proteins to ∼103-104 D for large proteins and complexes, but outliers in both directions exist. Dipoles with p J 400-600 D (pcrit) must be substantially oriented within the high-E half-cycle of the FAIMS waveform, with lesser alignment in the other halfcycle. Thus, the alignment of proteins above ∼20-30 kDa is likely, though some smaller proteins may align and some larger ones may not. The mobility of aligned ions is set not by the orientationally averaged cross-sections Ωavg but rather by collision integrals Ωp in the plane orthogonal to the dipole (with a weighted orientational averaging due to pendular motion). In general, that may either increase K at high E (promoting type A- or B-type behavior) or decrease it, favoring type C behavior. The effect on mobility may be dramatic (>15%), far outweighing the known decrease of K(E) for large ions due to other phenomena and thus possibly converting ions from type C to type A or B. Thus, the dipole alignment explains all key new observations from our study: the existence of abundant B-type ions for large proteins, their abrupt appearance above ∼25-30 kDa, and their extreme CVs. For CA II and ADH, the fraction of B-type conformers grows at higher charge states (z) and upon solution denaturation, increasing from ∼1-5% for the lowest measured z from pseudonative media to ∼10-50% for high z from the same and to 80-95% for high z from denaturing media (Figure 12). For BSA ions of any measured z value, the B-type fraction is ∼6080% with pseudonative media and a lower ∼45-60% with denaturing media. Quantitative interpretation of those data

Shvartsburg et al. requires detailed mobility calculations (including the dipole alignment) for representative optimized structures, an effort beyond the scope of this work. However, qualitatively the dipole alignment model is consistent with the above observations: Either (i) denatured dr-BSA conformers have an even higher p value compared with that of di-BSA resulting in dipole locking over both waveform half-cycles that would diminish the effect of alignment on CV or (ii) denaturation of helical domains destroys the tertiary structure thus generally improving the alignment of helices (and increasing p) and, in parallel, unwinds those into unstructured chains and loops (thus decreasing p). Conformers created by unfolding and refolding processes are exceptionally diverse even for cytochrome c74 and should be yet more so for larger proteins. Their p values certainly span a broad range that brackets the p value of the native conformation, pnat. Then denaturation would generally increase the fraction of conformers with p values above the threshold for dipole alignment (pcrit) when pnat < pcrit (as for CA II) and decrease that fraction when pnat > pcrit (as for BSA). The above constitutes circumstantial evidence for the dipole alignment hypothesis. However, regardless of whether the specific effects reported here are due to dipole alignment, the dipoles with moments exceeding ∼400 D must be substantially oriented at the highest E in FAIMS. At least some large protein ions indisputably have p exceeding that threshold (and by a large margin) and thus will be aligned at least during a part of the FAIMS cycle. That should strongly affect the mobility of such ions and hence their FAIMS separation properties. Future Directions. Future studies will explore FAIMS properties of additional proteins to better delineate the prevalence and onset of the newly found B-type behavior for large proteins and other macroions. This phenomenon and its tentative explanation in terms of dipole alignment suggest several exciting research directions. First, one could assess the dipole moments of macroions by measuring the extent of alignment as a function of peak FAIMS voltage and/or gas temperature. Second, absolute cross-sections orthogonal to the dipoles could potentially be obtained by knowing (Ωp - Ωavg) from FAIMS and Ωavg from IMS. Comparing the result with Ωp computed as outlined here for plausible candidate geometries could reveal new information about the ion structure. An analogous procedure, matching the measured Ωavg with calculations employing eq 6, has provided numerous insights into the structure and isomerization thermodynamics and kinetics of diverse gas-phase systems including atomic9,65,66,88-90 and molecular91 nanoclusters, peptides,92 organic polymers,93 oligonucleotides,94 oligosaccharides,95 and proteins.73,74 However, multiple isomers often have similar predicted Ωavg values, making IMS insufficiently specific for accurate structural elucidation. Measured p, Ωp, and Ωavg values for the same ion would allow a much more specific and reliable structural characterization than just the Ωavg value available from IMS. From an analytical perspective, dipole alignment expands the FAIMS separation space (at any z value) by an order of magnitude, from ∼2-8 V/mm for small proteins with type C ions only (Figures 5-8) to >40 V/mm for large proteins with type B and C ions (Figures 9-11). This greatly increases the separation peak capacity, especially for isomeric separations. For example, FAIMS devices operated at high resolution may separate protein conformers with the EC difference of ∼0.30.4 V/mm.55,57 Under those conditions, the maximum FAIMS peak capacity for proteins would exceed 100 in the presence of dipole alignment versus ∼5-20 in its absence. (Presently obtained values for both are approximately 4 times smaller

FAIMS Studies of Proteins because of relatively low FAIMS resolution.) The 2-D peak capacity and specificity of FAIMS/IMS are much superior to those of FAIMS alone,42 and new conformers for ubiquitin and cytochrome c (exhibiting no dipole alignment) were distinguished.57 Coupling to IMS should similarly augment the separation power of FAIMS under dipole alignment conditions, creating a maximum 2-D peak capacity of ∼103 for protein conformers. Considering the advantages of ion dipole alignment for FAIMS analyses, its extension to weaker dipoles is of interest. By eq 8, the paths to that are stronger dispersion fields and/or cooling the carrier gas. With N2 gas at STP, raising absolute Emax in Selectra much over 3 kV/mm (i.e., by >20% above 2.5 kV/mm used here) will cause an electrical breakdown across the gap,96 making FAIMS inoperable. Narrower gaps permit somewhat higher Emax values, e.g., ∼4-5 kV/mm for the 0.5 mm gap96 adopted in miniaturized FAIMS.20,22,47,48 Those Emax values will allow reduction of the pcrit value from its present value of ∼400 to ∼200-250 D. More insulating carrier gases would allow much lower pcrit. For example, the breakdown voltage of SF6 (at STP) over a 0.5-2 mm gap exceeds that for air or N2 ∼2-fold,96 which means Emax ≈ 8-9 kV/mm and pcrit ≈ 110-130 D. That would cover almost all protein ions (in near-native conformations). Still lower pcrit values could be achieved at higher pressures and/or lower temperatures. For example, cooling the gas from room T to 100 K would reduce pcrit 3-fold, from ∼250-400 D (depending on the gap width) to ∼80-130 D, again covering almost all proteins. A higher gas number density at lower T permits increasing Emax and thus reducing pcrit yet further to

Field Asymmetric Waveform Ion Mobility Spectrometry Studies of

Recommend Documents