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Mobility Measurement of Non- Denatured Protein and Protein Cluster Ions by DMA-MS Christopher J. Hogan 1 & Juan Fernandez de la Mora 2 1 Mechanical Engineering, University of Minnesota 2 Mechanical Engineering, Yale University Outline


  1. Mobility Measurement of Non- Denatured Protein and Protein Cluster Ions by DMA-MS Christopher J. Hogan 1 & Juan Fernandez de la Mora 2 1 Mechanical Engineering, University of Minnesota 2 Mechanical Engineering, Yale University

  2. Outline • Introduction & Methods – Motivation – Operating principle of DMA-MS – Study of charge-reduced protein and protein cluster ions • Results – Tandem mobility-mass plots – Inferring protein “size” from shape – Comparison to GEMMA results – Comparison to drift-tube IMS-MS • Summary & Conclusions

  3. Motivation • Goal of gas-phase mobility measurement: – Infer protein/complex structure or changes to structure in solution. – Electrospray under non-denaturing conditions – Maintain structural integrity prior to and during mobility measurement • Drift tube IMS or SYNAPT HDMS: – Often several high field regions Source: Shelimov et al., 1997 Source: www.waters.com

  4. Differential Mobility Analyzer • Parallel Plate DMA SEADM Electrospray DMA P4 Ionization – Spatial Mobility Filter Chamber • Constant Stream Monomobile Ions Separation – Separation at Atmospheric Region To MS Pressure – Good Ion Transmission (> Sheath Velocity, U 50% of selected mobility) δ 2 U = Z – High Resolving Power Inlet p LV DMA • SEADM DMA P3: R > 70 V I V O + L • SEADM DMA P4: R ~ 50 Z p : ion mobility Selected Outlet – Can be installed on the Ion V DMA = V I - V O front end of commercial δ mass spectrometer

  5. DMA-MS • DMA P4 coupled to QSTAR XL (Sciex) – Enables measurement of tandem mobility-mass spectra in a wide mobility and mass range (up to 40,000 in m/z). – Used with ESI source System Blower Control Box DMA

  6. Goals • Electrospray globular protein & protein cluster ions under non-denaturating conditions • From tandem mass-mobility spectra, infer protein sizes – Accounting for surface roughness and diffuse collisions – Compare to: • GEMMA (Gas-phase electrophoretic macromolecular mobility analyzer) • Drift tube IMS-MS data

  7. Electrospray Ionization • 40 µ m I.D., 360 µ m O. D. capillary • Use of charge reducing buffer triethylammonium formate. • Mitigates Coulombic stretching as well as polarization influences (Air, 8.7 times more polarizable than He) With Triethylammonium + With Ammonium Acetate + From Hogan et al. 2009

  8. DMA Calibration • DMA sheath flow (> 100 l min -1 ) must be R > 50 determined. – Simpler to measure mobility of a standard ion – DMA is a linear spectrometer, with the voltages applied measurement is made in (Tetraheptylammonium-Bromide) 2 the low-field limit. Tetraheptylammonium + – Calibrant mobilities known in air at 20 o C (Ude and – Adjust mobility assuming Fernandez de la Mora, ion is ~hard sphere. 2005). – DMA runs at 30 o C. Z V Z = s s – Choose largest singly charged calibrant possible V DMA

  9. Results • Protein concentrations of ~10-30 µ M used. • Results in formation of protein cluster ions. • Range: Cytochrome C momoners (12.2 kDa) to Concanavalin A hexamers (~150 kDa) • Mobility measurement made before any Lysosyme Ions, declustering no declustering • Declustering aids in sharpening mass peaks • Residual solute possibly bound to protein ions (increases peak FWHM) • Declustering promotes charge loss between the DMA and MS. • Fragmentation of multimer ions minimal • All observed peaks can be attributed to a specific multimer ion with a specific charge state with declustering

  10. Results • Fragmentation is observed for GroEL 14-mers

  11. Results

  12. Results

  13. Coulombic Stretching & Polarization Ζ • Hard sphere ze k T 9 = ( ) B mobility equation: π + + πα + 2 8 m 1 m / m p ( 1 / 8 ) d d g g i I i g • Hard sphere mobility equation- Ω = π/4 (d i +d g ) 2 (1+ πα I /8) • (z/Z) 1/2 ~ Ω 1/2 (Length scale) • For compact ions, without Coulombic stretching and polarization influences (z/Z) 1/2 ~ m 1/3 (m: ion mass) • Linear relationship implies minimal polarization effect/ Coulombic stretching • Measured ions are reasonably compact

  14. Coulombic Stretching & Polarization • Without charge reducing buffer, effects of Coulombic stretching and polarization observed (still a small effect with charge reducing buffer) GroEL 14 mers, Electrosprayed in NH 4 Ac buffer

  15. Inferring Cross-Sections/ Diameters Ζ k T ze 9 = B ( ) π + + πα + 2 8 m 1 m / m p ( 1 / 8 ) d d g g i I i g Momentum Accommodation coefficient 4Ω/π In Air (and N 2 ), known to be 0.91 1,2 . 1. Davies, C. N., Definitive equations for the fluid resistance of spheres. Proceedings of the Physical Society 1945 , 57, 259-270. 2. Allen, M. D.; Raabe, O. G., Re-evaluation of Millikan's oil drop data for the motion of small particles in air. Journal of Aerosol Science 1982 , 13, 537. d g : bath gas diameter d i : ion “diameter”, independent of bath gas. For sufficiently large spherical ions, d i is the volume diameter Ω : Collision cross-section. Gas dependent, but best used for model (EHSS) EMI-BF 4 cluster measurement from comparisons (if the model is fine enough to Larriba et al., in prep capture multiple scattering events)

  16. Inferring Cross-Sections/ Diameters Ζ ze 9 k T = B ( ) π + + πα + 2 8 m 1 m / m p ( 1 / 8 ) d d g g i I i g • Solid line: Predicted curve with α I = 0.91, using known volumes from Larriba et al., in prep of cation and anion as well as known volume fraction. • Dashed line: Polarization Limit Doubly Charged • Excellent agreement (<1% difference between predictions and measurements) Singly Charged • We therefore use α I = 0.91, d g = 0.3nm in inferring d i from Z, z, measurements

  17. Comparison to GEMMA Results • Prior DMA based protein measurements (pioneered by Stan Kaufman) – Singly charged protein ions (5 kDa to several MDa) • Black circles- This study • White Squares- Kaufman et al., 1996 • Grey Triangles- Bacher et al., 2001 and Kaddis et al., 2007. • Diameter ~ Mass 1/3 • Kaufman Density: 0.89 g cm -3 • Difference with Kaufman et al: attributable to solute adducts • Bacher+Kaddis Density: • Bulk peptide density: 1.35 g cm -3 0.67 g cm -3 • Kaddis+Bacher Denisty: Low • This Study: 0.95 g cm -3

  18. Comparison to Drift tube Results • Drift tube Measurements: – Often used as standards for T-WAVE calibration (need to be extremely reliable) – Made in He (~d g = 0.2 nm) – Made from electrosprayed ions in acidic solution (or MALDI) – Comparison of equivalent charge state ions (in terms of ion diameter): • Lysozyme +5 : – Clemmer and coworkers: 3.89 nm ( α I =0), 3.37 nm ( α I = 0.91) – This study: 4.01-4.18 nm ( α I =0), 3.40-3.54 nm ( α I = 0.91) – Fernandez-Lima et al. (2010, MALDI, +1 ion): 3.40 nm ( α I =0), 2.89 ( α I = 0.91) • Cytochrome C +4 – Clemmer and coworkers: 3.63 nm ( α I =0), 3.09 nm ( α I = 0.91) – This study: 3.85-4.00 nm ( α I =0), 3.26-3.39 nm ( α I = 0.91) – Fernandez-Lima et al. (2010, MALDI, +1 ion): 3.34 nm ( α I =0), 2.84 ( α I = 0.91)

  19. Summary & Conclusions • DMA-MS can be successfully employed to measure the m/z and mobility of non-denatured electrospray- generated protein ions • Charge reducing buffer mitigates the effects of Coulombic stretching and polarization • Mobility measurements made immediately following droplet evaporation • Despite solute clustering onto ions during measurements, relatively compact ions are found – Ion sizes inferred with α I = 0.91 – Suggest further investigation of α I in He for d i determination. • Future work: further interpreting protein structure from DMA-MS measurements – GroEL: Partially collapsed gas-phase structure observed

  20. Acknowledgements • DMA P4- supplied by SEADM, Boecillo, Spain • QSTAR-XL Provided by Applied Biosystems • Laboratory space provided by the Keck Biotechnology Center • We thank Brandon Ruotolo, Joe Loo, and Bruce Andrien for visiting Yale University and providing unique protein samples to examine (most data not shown)

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