Ion Mobility Spectrometry of Macromolecules with Dipole Alignment

20 hours ago - Since inception in 1980-s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) was implemented at or near the a...
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Ion Mobility Spectrometry of Macromolecules with Dipole Alignment Switchable by Varying the Gas Pressure Alexandre A. Shvartsburg, Roch Andrzejewski, Andrew Entwistle, and Roger Giles Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00525 • Publication Date (Web): 05 Jun 2019 Downloaded from http://pubs.acs.org on June 6, 2019

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Ion Mobility Spectrometry of Macromolecules with Dipole Alignment Switchable by Varying the Gas Pressure Alexandre A. Shvartsburg,1,* Roch Andrzejewski,2 Andrew Entwistle,2 Roger Giles2 1. Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260, United States 2. Shimadzu Research Laboratory, Wharfside, Trafford Wharf Road, Manchester M17 1GP, United Kingdom * [email protected] ABSTRACT: Since inception in 1980-s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) was implemented at or near the ambient gas pressure (AP). Recently we developed FAIMS at 15 - 30 Torr within a mass spectrometer and demonstrated it for small and medium-size ions, including peptides. The overall separation properties mirrored those at AP, reflecting the shared underlying physics. Here we extend these analyses to macromolecules, namely multiply-charged proteins generated by electrospray ionization. The spectra for smaller proteins (ubiquitin, cytochrome c, myoglobin) again resemble those at AP, producing features for one or few adjacent well-defined conformers with type C behavior. Large proteins (single aldolase domain and albumin) now follow, with no broad bands for type A or B species that dominated at 1 Atm. Those unique behaviors were ascribed to pendular ions with electric dipoles reversibly locked by strong field in FAIMS. Disappearance of those bands shows loss of alignment predicted by firstprinciples theory, further supporting dipole locking at AP. The capability to modulate dipole alignment by varying gas pressure at constant normalized field provides the basis for determining the ion dipole moment and direction within molecular frame from the pressure of onset and characteristics of spectral drift. This new approach to alter FAIMS separations of proteins could make a powerful tool for structural biology and be useful for proteomics and imaging.

Ion mobility spectrometry (IMS) can separate and characterize charged species based on the properties of their transport through gases driven by electric field of some intensity (E).1-4 In the classic drift-tube (DT) IMS implementation, a moderate fixed E is established over known distance inside a gas-filled enclosure.1-3 Ions travel through it at constant velocity v that is proportional to E over the gas number density (N): v = KE = K0 N0 E/N (1) Here, N0 is N under standard temperature and pressure (2.687×1025 m−3), K is mobility, and K0 is K at STP - the descriptor of ion/gas molecule pair set by their identities and gas temperature (T).1,2 As ions are normally free to rotate, K0 is determined2 by the ion-molecule collision cross section averaged over all partner orientations (ΩAVG). Other pertinent properties, such as the magnitude of ion heating by above-thermal collisions2,4 and threshold for electrical breakdown in gas,5 are also governed by E/N expressed2 in Townsend (1 Td = 1×10−21 V×m2). Subsequent IMS configurations utilized a dynamic field in stationary gas (traveling-wave6,7 and transverse-modulation8 IMS) or static field in moving gas (aspiration9 or trapped10 IMS). These linear IMS methods separate ions by K0 at moderate E/N, where K0 does not materially depend2,4 on E/N and v is proportional to E/N by eq (1). Coupling to mass spectrometry (MS) in 1960-s opened the door to practical IMS applications such as isomeric analyses of aromatics.11 Emergence of soft ionization sources in 1990-s revolutionized IMS/MS, extending it to proteomic, metabolomic, petroleomic, forensic, and environmental

analyses.12-15 The ability to extract ion geometries from IMS data by matching measured ΩAVG with molecular dynamics calculations brought IMS to nanoscience and structural biology.16,17 Recent commercialization of diverse IMS/MS platforms expanded the technology to broad user community.6,10,18 Huge gains of resolving power in designs employing a dynamic field19 and/or gas flow10 permitted separating structures with 400 D prevailing40

Figure 1. Total FAIMS spectra (top) and EC/charge state diagrams for myoglobin (a) and albumin (b). Reproduced from Ref. 39. Copyright 2006 American Chemical Society. above ~30 kDa. In fact, the carbonic anhydrase II (29 kDa), alcohol dehydrogenaze (Ald, 44 kDa), bovine serum albumin (Alb, 66.7 kDa), and transferrin (Tr, 78 kDa) exhibited (Figure 1) major “type A” peaks at EC 2000 were not measurable. As same applied at AP,39,40,44,46 the results directly compare.

Figure 3. Measured protein mass spectra. Charge states are labeled, those selected for FAIMS analyses are in red.

Heating and survival of intact proteins at extreme field We acquired the FAIMS spectra for lowest, intermediate, and (except for Cyt) highest sufficiently intense charge states (Figure 3) with quadrupole in the selected ion monitoring (SIM) mode (Figures 4, 5, and S1 - S3). In all cases, the signal with s/n > 10 persisted to at least ED/N = 250 Td - about the maximum in prior LP-FAIMS31 or microchips.45,51 The lower charge states of all proteins exhibited enough signal up to the breakdown threshold. Assuming K(0) = 1.0 cm2/(Vs) measured38 for unfolded Ub conformers (z = 10 -

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EC/N, Td Figure 4. The EC/ED palettes for ubiquitin with z = 6, 10, 13. The vertically scaled FAIMS spectra at highest ED with good signal (marked by arrows in palettes) are on top, with peak widths and resolving power values listed. The scaling factors convey peak heights relative to the charge state with greatest signal (here 10+). The EC(ED) curve bend areas are in white rectangles. With any protein, the signal decay at greater ED was faster for higher charge states. Except with the largest protein Alb (Figure 5), the data for highest z end short of the breakdown field (Figures 4, S1 - S3). For example, the maximum useful ED/N decreases from 284 Td for Ub 6+, Myo 9+, or Ald 36+ to ~260 Td for Ub 13+, Myo 26+, or Ald 56+ (Figures 4, S2, S3). The practical limitation of ED/N range by ion loss seen here may be caused by (i) dissociation promoted by stronger field heating, (ii) "self-cleaning" of isomerized heated ions upon EC move beyond the peak width, wherein the altered species are removed by FAIMS action,38,57 (iii) faster diffusion of hotter ions, especially its anisotropic component along the field (i.e., across the gap)4,52 (iv) effective gap narrowing with increasing

oscillation amplitude in the cycle (about proportional to ED/N).52,53 The interplay of these factors in LP-FAIMS of proteins will be discussed in a future publication. In summary, macroions can broadly endure FAIMS process at fields up to ~300 Td for >10 ms. This new regime ought to allow many novel analyses. Here we focus on the crucial dipole alignment aspect.

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ECFAIMS /N, Tdseparations showing no alignment Figure 5. Same as Fig. 4 for albumin with z = 36, 47, 55.

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The palettes for all proteins (at any z) are now analogous: the EC(ED) functions smoothly increase up to the maximum ED with positive 2nd derivative (Figures 4, 5, S1 - S3). This type C behavior, where K continually decreases at higher E/N, is classic for peptides and non-aligned small proteins at AP24,26,39,40,43,46,55 and peptides at low pressure.31 No signal was found at EC < 0 or (at relevant ED) EC ~ 0. These findings copy those at AP for small proteins (e.g., Figure 1a), but totally differ from those for Alb (Figure 1b). Further, the curves lie close across proteins. At the maximum ED/N, all come at EC/N = 4.5 - 6 Td and 5 - 8 Td for the lowest and highest z, respectively. The difference between the curves for Alb and mean trend for small proteins (not exhibiting alignment at AP) is less than the scatter around trend for those proteins. For example, the EC/N for Ub (5.5 Td), Cyt (5.4 Td, mean of two conformers), and Myo (4.6 Td) differ by 0.9 Td, but average to same 5.2 Td as EC/N for Alb at lowest z. The peak widths for Alb are likewise similar to those for small proteins. The w for highest z is 0.4 Td, same as the mean for Ub (0.4 Td), Myo (0.3 Td), and Ald (0.6 Td). The mean w over all charge states increases from 0.4 Td for Ub to 0.6 Td for Myo, but not further to Ald and Alb. In contrast, at AP39,40 the band abruptly expands by >10-fold around 30 kDa. The instrumental peak widths in planar-gap FAIMS scale52,53 approximately as K−1/2. As larger protein ions are somewhat less mobile despite higher z, a modest gradual peak broadening is expected and does not suggest alignment or structural heterogeneity.41 Like for small proteins at AP, the peak widths here narrow at higher z: on average, from 0.7 Td for the lowest z to 0.5 Td for highest z. This presumably happens as complete unfolding reduces polymorphism.38,39,44 To the contrary, the bands for aligned proteins did not narrow at higher z, but often got wider with firmer alignment.39,40 All these findings indicate no alignment in LP-FAIMS and, conversely, confirm that of large proteins in AP-FAIMS.39-41,46 At AP, the EC values maximized in lower z for small proteins38,55,57 (e.g., 7+ or 8+ for Ub and 9+ for Myo, Figure 1a) and remained near-constant for large proteins39,40 (e.g., Alb, Figure 1b). That was due to swift EC drop upon unfolding at intermediate z, only partly offset by EC increase at higher z for

EC/N, Td Figure 6. Selected FAIMS spectra showing conformer resolution (parameters labeled as in Figures 4, 5).

fixed geometries.38,57,59 Here, a substantial unfolding already at lowest z (induced by more severe field heating) apparently leaves less room for unfolding over the probed z range. We still see structural transitions and co-existing distinct conformers. For Ub and Myo with lowest z, the EC(ED) curves kink toward lower EC at ED/N ~ 200 Td (Figures 4, S2). For Ub with highest z and Myo and (prominently) Cyt with lowest z (Figures S1, S2), two conformers appear in same ED/N range and last to the breakdown (Figure 6). The lack of multiple peaks or curve inflections for Ald and Alb is common for large proteins, where many similar conformers allow less discrete transitions through pathways with more intermediates.39,40 The curve bending to lower EC may suggest unfolding.38,57 However, the coexistence of conformers at maximum ED/N, modest EC difference between them, and their occurrence for highest z (fully unfolded even for much colder ions in APFAIMS or linear IMS with no field heating)55 all point to the multiple unfolded conformers discovered by AP-FAIMS.44,55 Their origin is uncertain, but feasibly manifests protomers (peptides with alternative protonated residues) that would interconvert less readily than conformers within a protonation scheme.55 Anyhow, not merely proteins but specific conformers broadly survive to at least 284 Td despite peak heating to ~600 oC. At R > 100 in AP-FAIMS, smaller proteins (Ub, Cyt, Myo) with high z featured ~10 distinct peaks with widths close to those for peptides and thus corresponding to essentially single conformers. The widths for Ub13+ and Myo26+ here (0.3 - 0.4 Td, Figures 4 and S2) only slightly exceed w = 0.2 - 0.3 Td for peptides at same 15 Torr.31 This reveals no hidden structural heterogeneity (considering the above-mentioned trivial peak broadening for larger proteins),41 but does not prove its absence. This situation mirrors AP-FAIMS with similar lower resolving power of 10 - 20, where the peak width is controlled mostly by diffusion instead of conformational multiplicity.38,55

Conclusions and Future Projections We report first analyses of macromolecules employing the recently engineered low-pressure FAIMS (integrated with mass spectrometry).31 Separations of small proteins, peptides, amino acids, and other small ions track the AP benchmarks. The behavior of large proteins drastically differs: there is no signal at CV < 0 for species with mobility rising in stronger fields. This dichotomy reflects the locking of electric macrodipoles of large proteins at ambient but not low pressure, while smaller species with weaker dipoles are free rotors in either case. These findings accord with a priori theory for dipole alignment of ions in gases, requiring (at any field) a minimum dipole moment p proportional to the inverse gas pressure.39-41,46 Then alignment in low-pressure FAIMS would occur for p over ~104 D projected for MDa-size biomolecules. Present results further prove the dipole locking in high-field IMS and enable characterizing the dipoles for isolated macromolecules by tracking the pressure-dependent evolution of nonlinear ion mobility spectra. The point for onset and characteristics of spectral drift would reveal the dipole moment and orientation in the ion, respectively. Given the exceptional structural specificity of dipole magnitude and direction within the molecular frame, a path to deduce them opened by present work is anticipated to benefit integrative structural biology. The option to modify FAIMS spectra by tuning the dipole alignment

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Analytical Chemistry may also facilitate (i) advancing proteoform separations24,26 to larger proteins and top-down workflows, and (ii) use of FAIMS in tissue imaging, where pulling the pendular large proteins apart from small proteins and other species alleviates spectral congestion and increases the number of identified proteins.60-62 The linear IMS spectra for macromolecules are now routinely annotated in terms of structures.17,63 This is achieved by relating the measured mobilities to those computed for optimized ion geometries via first-principle models suitable at low field.2,64,65 Modeling high-field ion transport is tremendously more challenging,2,4 and no parallel procedure for polyatomic ions currently exists. We hope these findings stimulate the development of high-field ion mobility calculations, in particular for macromolecules.

ACKNOWLEDGMENT The work at WSU was supported by NSF CAREER Award (CHE1552640). The corresponding author is a consultant to Shimadzu Research Laboratory.

Supporting Information Available The EC/ED palettes for Cytochrome c, Myoglobin, and Aldolase..

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