Development of an Ion Mobility Quadrupole Time of Flight Mass

Anal. Chem. 2008, 80, 6336–6344

Development of an Ion Mobility Quadrupole Time of Flight Mass Spectrometer Bryan J. McCullough,† Jason Kalapothakis, Hayden Eastwood, Paul Kemper,‡ Derek MacMillan,§ Karen Taylor,| Julia Dorin,| and Perdita E. Barran* School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, U.K. We describe here a new ion mobility capable mass spectrometer which comprises a drift cell for mobility separation and a quadrapole time of flight mass spectrometer for mass analysissthe MoQTOF. A commercial QToF instrument (Micromass UK Ltd., Manchester, UK) has been modified by the inclusion of an additional chamber containing a drift cell and ancillary ion optics. The drift cell is 5.1 cm long made from a copper block and is mounted from a top hat flange in a chamber situated post source optics and prior to the quadapole analyzer. Details of this instrument are provided along with information about how it can be used to acquire mobilities of ions along with their mass to charge ratios. The MoQTOF is used to examine conformations of a series of antimicrobial peptides based on a β-defensin template. In vivo, these cationic cystine-rich amphiphilic peptides are conformationally restrained by three or more disulfide bridges, although recent findings by several groups have cast doubt on the importance of canonical disulfide pairing to antimicrobial activities. By synthesizing a panel of variants to Defb14 (the murine orthologue of HBD3), we exploit ion mobility to distinguish conformational differences which arise due to disulfide formation and to the hydrophobicity of the peptide sequence. Our gasphase results are interpreted in terms of the antimicrobial and chemotacic properties of β-defensins, and this mass spectrometry based approach to discern structure may have a role in future design of novel antibiotics. The analytical technique of ion mobility spectrometry was developed by Cohen and Karasek in 1970 as a sensor1 building on earlier gas-phase ion chemistry investigations.2,3 It has since been used to detect a wide range of analytes including illegal drugs, chemical warfare agents, explosives, and environmental * Corresponding author. E-mail: [email protected]. † Present address: The Michael Barber Centre for Mass Spectrometry, The University of Manchester, MIB, 131 Princess Street, Manchester, M1 7DN, U.K. ‡ Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106. § Department of Chemistry, University College London, 20 Gordon Street London, WC1H 0AJ, U.K. | MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, U.K. (1) Cohen, M. J.; Karasek, F. W. J. Chromatogr. Sci. 1970, 8, 330. (2) George, G.; Stevenson, D. P. J. Chem. Phys. 1958, 29, 294–299. (3) Mason, E. A.; Mc Daniel, E. W. Transport Properties of Ions in Gases; Wiley: New York, 1988.

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Analytical Chemistry, Vol. 80, No. 16, August 15, 2008

pollutants.4 Ion mobility is a measure of how quickly a gas phase ion moves through a buffer gas under the influence of an electric field, and this depends on two factors: the rotationally averaged collision cross section of the ion and the charge present on it. By measuring the drift time of an ion through a known distance, it is possible to determine its collision cross section with some degree of accuracy. In instruments where the drift field is a dc potential, the relationship between the collision cross section of an ion (Ω) and the measured average drift time (tD) is given by

Ω)

[

1 (18π)1/2 ze 1 + 16 (k T)1/2 mI mB B

]

1/2 tDE 760

L

T 1 P 273.2 N

(1)

where z is the charge state of the ion; mI is the mass of the ion and mB of the buffer gas; E is the electric field; L is the length of the drift tube; P and T are the pressure and temperature of the buffer gas; and N is the neutral number density. This version of the equation contains the expression for the reduced mobility. The experimental collision cross section can be compared to cross sections predicted from coordinates obtained from other structural investigations or from computational measurements to obtain atomistically detailed conformational information. In recent years, ion mobility coupled with mass spectrometry (IM-MS) has gained importance as a tool for structural analysis and particularly for its use to reveal the conformation of biological molecules. After developments in soft ionization methods, IM-MS studies of biologically relevant species started in the mid and late 1990s on home-built instruments which coupled two well-known analytical techniques. Some of the most influential work in this period was performed by Bowers,5–10 Jarrold,11–14 Clemmer,15–19 (4) Hill, H. H.; Siems, W. F.; Stlouis, R. H.; Mcminn, D. G. Anal. Chem. 1990, 62, A1201–A1209. (5) Wyttenbach, T.; Witt, M.; Bowers, M. T. J. Am. Chem. Soc. 2000, 122, 3458–3464. (6) Gill, A. C.; Jennings, K. R.; Wyttenbach, T.; Bowers, M. T. Int. J. Mass Spectrom. 2000, 195, 685–697. (7) Wyttenbach, T.; Batka, J J., Jr.; Gidden, J.; Bowers, M. T. Int. J. Mass Spectrom. 1999, 193, 143–152. (8) von Helden, G.; Wyttenbach, T.; Bowers, M. T. Science 1995, 267, 1483– 1485. (9) Wyttenbach, T.; Bushnell, J. E.; Bowers, M. T. J. Am. Chem. Soc. 1998, 120, 5098–5103. (10) Bowers, M. T.; Wyttenbach, T.; Gidden, J. Book of Abstracts, 215th ACS National Meeting, Dallas, March 29-April 2 1998, ANYL-116. (11) Shvartsburg, A. A.; Schatz, G. C.; Jarrold, M. F. J. Chem. Phys. 1998, 108, 2416–2423. (12) Hudgins, R. R.; Ratner, M. A.; Jarrold, M. F. J. Am. Chem. Soc. 1998, 120, 12974–12975. (13) Shelimov, K. B.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2987–2994. 10.1021/ac800651b CCC: $40.75  2008 American Chemical Society Published on Web 07/15/2008

and Hill,20–23 and their investigations have paved the way for others and prompted development of commercially available mobility devices, as the power of this technique for biological analysis became apparent. IM-MS has now been successfully employed to provide insight to several key areas of structural biology. Since an early seminal study on the protein Cytochrome C in 1997,14 Clemmer and co-workers have used IM-MS to examine the unfolding dynamics of proteins as a function of charge state and temperature24–26 using IM-MS devices of increasing sophistication in terms of their resolving power.27–29 Bowers and co-workers have made significant progress employing IM-MS coupled with molecular modeling to study systems of direct biological relevance.30–32 Of particular note are their recent findings on the effects of amyloid peptide aggregation which point to an understanding of the early stages of Alzheimer’s and other neurodegenerative diseases.33 Building from preliminary studies on diand oligo-nucleotides,30 the same group has also made several detailed investigations of the conformations adopted by telomeric DNA sequences as isolated gas-phase ions34,35 and also bound to stabilizing ligands;36 this latter study has implications for the design of anticancer agents which are required to stabilize a given promoter or telomeric region of DNA. As well as its use for structural characterization, ion mobility is gaining in popularity (14) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240–2248. (15) Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141–10142. (16) Hoaglund, C. S.; Liu, Y. S.; Ellington, A. D.; Pagel, M.; Clemmer, D. E. J. Am. Chem. Soc. 1997, 119, 9051–9052. (17) Liu, Y. S.; Clemmer, D. E. Anal. Chem. 1997, 69, 2504–2509. (18) Valentine, S. J.; Anderson, J. G.; Ellington, A. D.; Clemmer, D. E. J. Phys. Chem. B 1997, 101, 3891–3900. (19) Hoaglund, C. S.; Valentine, S. J.; Sporleder, C. R.; Reilly, J. P.; Clemmer, D. E. Anal. Chem. 1998, 70, 2236–2242. (20) Wu, C.; Klasmeier, J.; Hill, H. H. Rapid Commun. Mass Spectrom. 1999, 13, 1138–1142. (21) Wu, C.; Siems, W. F.; Asbury, G. R.; Hill, H. H. Anal. Chem. 1998, 70, 4929–4938. (22) Lee, D. S.; Wu, C.; Hill, H. H. J. Chromatogr. A 1998, 822, 1–9. (23) Wittmer, D.; Luckenbill, B. K.; Hill, H. H.; Chen, Y. H. Anal. Chem. 1994, 66, 2348–2355. (24) Badman, E. R.; Hoaglund-Hyzer, C. S.; Clemmer, D. E. Anal. Chem. 2001, 73, 6000–6007. (25) Myung, S.; Badman, E. R.; Lee, Y. J.; Clemmer, D. E. J. Phys. Chem. A 2002, 106, 9976–9982. (26) Valentine, S. J.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 2002, 13, 506– 517. (27) Valentine, S. J.; Koeniger, S. L.; Clemmer, D. E. Anal. Chem. 2003, 75, 6202–6208. (28) Koeniger, S. L.; Valentine, S. J.; Myung, S.; Plasencia, M.; Lee, Y. J.; Clemmer, D. E. J. Proteome Res. 2005, 4, 25–35. (29) Koeniger, S. L.; Merenbloom, S. I.; Valentine, S. J.; Jarrold, M. F.; Udseth, H. R.; Smith, R. D.; Clemmer, D. E. Anal. Chem. 2006, 78, 4161–4174. (30) Gidden, J.; Ferzoco, A.; Baker, E. S.; Bowers, M. T. J. Am. Chem. Soc. 2004, 126, 15132–15140. (31) Bernstein, S. L.; Liu, D. F.; Wyttenbach, T.; Bowers, M. T.; Lee, J. C.; Gray, H. B.; Winkler, J. R. J. Am. Soc. Mass Spectrom. 2004, 15, 1435–1443. (32) Wyttenbach, T.; Bowers, M. T. Modern Mass Spectrometry, Topics In Current Chemistry; 2003; Vol. 225, pp 207-232. (33) Teplow, D. B.; Lazo, N. D.; Bitan, G.; Bernstein, S.; Wyttenbach, T.; Bowers, M. T.; Baumketner, A.; Shea, J. E.; Urbanc, B.; Cruz, L.; Borreguero, J.; Stanley, H. E. Acc. Chem. Res. 2006, 39, 635–645. (34) Baker, E. S.; Bernstein, S. L.; Gabelica, V.; De Pauw, E.; Bowers, M. T. Int. J. Mass Spectrom. 2006, 253, 225–237. (35) Baker, E. S.; Hong, J. W.; Gaylord, B. S.; Bazan, G. C.; Bowers, M. T. J. Am. Chem. Soc. 2006, 128, 8484–8492. (36) Gabelica, V.; ShammelBaker, E.; Teulade-Fichou, M. P.; DePauw, E.; Bowers, M. T. J. Am. Chem. Soc. 2007, 129, 895–904.

as a separation device for proteomic investigations. Over the past 8 years, Clemmer has performed a number of investigations where IM-MS has been applied to ever larger proteomes beginning with a test system of a complete ubiquitin digest37 and building up to the most recent work on the proteomes of Drosophila.38 Smith and co-workers have also begun to employ IM-MS for proteomic investigations39 and have recently described the addition of funnelshaped ion guides within the mobility apparatus.40 These increase the transmission efficiency, allowing longer drift tubes and yielding higher mobility resolution. Due in part to the potential of ion mobility mass spectrometry as a “separator” in two dimensions for complex mixtures, commercial IM-MS devices have become available in the past 5 years. Efforts in commercial development have concentrated on improving the low duty cycle that is inherent to linear ion mobility experiments and also on increasing the resolution available. Initial studies employing these devices have been applied as much to structural measurements as to proteome analysis, reflecting the growing power of mass spectrometry as a tool which provides detailed conformational information on biological moieties.41 In 1999, Purves et al.42 first reported the use of a high-field asymmetric waveform ion mobility spectrometry device (FAIMS) coupled to a mass spectrometer. In a FAIMS instrument, separation is achieved by ions moving in the presence of a gas under the influence of electric fields which are applied using a highfrequency periodic asymmetric waveform, rather than a dc voltage. As a consequence, the relationship between the mobility of an ion under these conditions and its collision cross section is not as clear-cut as that found in a linear instrument (as given by eq 1 above). Waters MS Technologies (Manchester, UK) recently introduced the first commercially available integrated IM-MS instrument, the Synapt HDMS.43,44 The RF applied to consecutive electrodes in the stacked ring ion guide within the ion mobility separator provides a potential well which keeps the ions radially confined within the device. To propel the ions through the device, a traveling wave comprising a series of transient DC voltages is superimposed on top of the RF voltage, and hence this device is sometimes referred to as a Traveling Wave Ion Guide (TWIG). This voltage is applied sequentially to pairs of ring electrodes providing a potential which can push ions through the device. These commercial available devices have already been used to good effect. Ashcroft and co-workers used FAIMS coupled to a quadrapole time of flight instrument to examine conformers of (37) Valentine, S. J.; Counterman, A. E.; Hoaglund, C. S.; Reilly, J. P.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1998, 9, 1213–1216. (38) Xun, Z. Y.; Sowell, R. A.; Kaufman, T. C.; Clemmer, D. E. J. Proteome Res. 2007, 6, 348–357. (39) Tang, K.; Li, F.; Shvartsburg, A. A.; Strittmatter, E. F.; Smith, R. D. Anal. Chem. 2005, 77, 6381–6388. (40) Tang, K.; Shvartsburg, A. A.; Lee, H. N.; Prior, D. C.; Buschbach, M. A.; Li, F.; Tolmachev, A. V.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2005, 77, 3330–3339. (41) Benesch, J. L. P.; Ruotolo, B. T.; Simmons, D. A.; Robinson, C. V. Chem. Rev. 2007, 107, 3544–3567. (42) Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszczyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094–4105. (43) www.waters.com. (44) Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. Int. J. Mass Spectrom. 2007, 261, 1–12.


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β-microglobulin.45Using a TWIG-based system, Robinson et al. have assessed conformations of multimeric proteins46 and also the disassembly of complexes viewing the partial unfolding of monomer units while still retaining some of the integrity of the compelx.47 To properly rationalize experimental collision cross sections in terms of the possible structures of the gas-phase ions that they could represent for dynamic many atom systems, for example, unfolding proteins, requires considerable computational effort. This has led to the development of tools which provide the collision cross sections of theoretically modeled candidate geometries, which are then compared to experimental findings.11,32,46,48,49 We too have followed this synergistic marrying of theory and experiment.50–53 We have previously described the use of IMMS to study the conformations of a small, disulfide-constrained, antimicrobial peptide, DEFB107, in various charge states.50 Using a combination of the experimental measurements and molecular modeling of the peptide, we are able to demonstrate the influence of the disulfide bonds on the gas-phase conformations formed by the peptide, and a more developed version of this approach is presented here. In this article, we describe the modification of a commercial mass spectrometer, a Micromass Q-ToF I (Micromass UK Ltd., Manchester, UK), to include an ion mobility device prior to the first mass analyzer. The instrument was designed such that major functionalities of the original instrument including high sensitivity, good resolution, and its MS/MS capabilities are retained with the advantage of an ion mobility separation prior to MS measurement. The use of the instrument is described. We present here data in the application of the instrument to the study of a series of peptides related to the murine β-defensin Defb14. These peptides have been synthesized with differing numbers of disulfide bonds which affects the range of conformations they can adopt in the gas phase and also their biological activity. The spread in gas-phase conformations provides insight to the in vivo behavior of these peptides, and also here are used as a test system for the general applicability of IM-MS to determine topologies of disulfide restricted proteins. EXPERIMENTAL Instrumentation. Figure 1 is a schematic diagram of the instrument, the MoQToF (mobility quadrupole time of flight). The instrument has been modified from the original mass spectrometer (45) Borysik, A. J. H.; Read, P.; Little, D. R.; Bateman, R. H.; Radford, S. E.; Ashcroft, A. E. Rapid Commun. Mass Spectrom. 2004, 18, 2229–2234. (46) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658–1661. (47) Ruotolo, B. T.; Hyung, S. J.; Robinson, P. M.; Giles, K.; Bateman, R. H.; Robinson, C. V. Angew. Chem., Int. Ed. Engl. 2007, 46, 8001–8004. (48) Mao, Y.; Ratner, M. A.; Jarrold, M. F. J. Am. Chem. Soc. 2001, 123, 6503– 6507. (49) Wyttenbach, T.; von Helden, G.; Batka, J. J., Jr.; Carlat, D.; Bowers, M. T. J. Am. Soc. Mass Spectrom. 1997, 8, 275–282. (50) McCullough, B. J.; Eastwood, H.; Clark, D. J.; Polfer, N. C.; Campopiano, D. J.; Dorin, J. A.; Maxwell, A.; Langley, R. J.; Govan, J. R. W.; Bernstein, S. L.; Bowers, M. T.; Barran, P. E. Int. J. Mass Spectrom. 2006, 252, 180– 188. (51) Jin, L.; Barran, P. E.; Deakin, J. A.; Lyon, M.; Uhrin, A. Phys. Chem. Chem. Phys. 2005, 7, 3464–3471. (52) Barran, P. E.; Roeseke, R. W.; Pawson, A. J.; Sellar, R.; Bowers, M. T.; Morgan, K.; Lu, Z. L.; Tsuda, M.; Kusakabe, T.; Millar, R. P. J. Biol. Chem. 2005, 280, 38569–38575. (53) Barran, P. E.; Polfer, N. C.; Campopiano, D. J.; Clarke, D. J.; LangridgeSmith, P. R. R.; Langley, R. J.; Govan, J. R. W.; Maxwell, A.; Dorin, J. R.; Millar, R. P.; Bowers, M. T. Int. J. Mass Spectrom. 2005, 240, 273–284.

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Figure 1. Schematic diagram of the MoQToF. (A) z-Spray ESI ion source; (B) vacuum chamber 1 housing precell hexapole; (C) vacuum chamber 2, new chamber housing the precell Einzel lens (D); drift cell (E); and postcell hexapole (F); this chamber also contains all gas and electric feedthroughs for the drift cell; (G) vacuum chamber 3 housing the quadrupole mass analyzer and hexapole collision cell leading to the orthogonal ToF mass analyzer (not shown).

by the inclusion of a vacuum chamber between the electrospray source and the quadrupole. This chamber, manufactured from an extruded aluminum block, houses the precell lens stack, drift cell, post cell lens, and a post cell hexapole and is pumped by a 500 ls-1 Pfeiffer TMH520 turbomolecular pump. Ions are produced in the ESI source and transferred through the source to a 20 cm hexapole in which they can be stored for mobility experiments. From here they are focused into the mobility cell using a three-element lens stack (with x/y steering on the central lens). Ions are stored in the hexapole by the application of a stopping voltage to the “top-hat” lens at the end of the hexapole. A reduction of the voltage on this lens gates the ions into the drift cell for mobility experiments. This is performed via a bipolar pulser power supply, and the triggering of this event starts our mobility clock (see below). We notice that with optimal lens settings and within a range of pressures we can store ions in the hexapole, thus minimizing the inevitable loss of ions that goes along with any IM-MS experiment. Upon exiting the drift cell, the ions are focused into the 12.5 cm postcell hexapole via a single lens. The ions are transmitted through this short hexapole into the next vacuum chamber which contains a quadrupole mass filter (which can be set to transmit or select an individual mass), a collision cell, and final transfer hexapole prior to the ToF. The ions are detected on a point detector (located at the top of the flight tube) when operating in quadrupole only mode and on an MCP detector in ToF mode. The signal from the MCP detector is converted via a 4 GHz TDC card into a total arrival time distribution (tATD) wherein the relative intensities of each ion are binned according to its drift time through the instrument. Figure 2 shows perspective cross sectional and sectional views of the drift cell used in the MoQToF. The cell is based on the design used by the Bowers group first reported in 1990.54 It is made from a copper block (65.5 mm × 88.9 mm × 88.9 mm) and copper end cap separated by a ceramic ring. The cell is suspended from a top hat flange which sits on the top of the drift cell chamber via four stainless steel rods, which are electrically insulated with ceramic breaks. The block (cell body) and end cap can be heated using a set of ceramic heaters and cooled with a stream of nitrogen. The temperature of each is monitored using a set of three thermocouples (1 on the body, 2 on the end cap). The pressure in the drift cell is measured via a 1/2 in. exit port on the top of the cell and recorded using a baratron (MKS). This 1/2 in. (54) Kemper, P. R.; Bowers, M. T. J. Am. Soc. Mass Spectrom. 1990, 1, 197– 207.

Figure 2. Cell drawings for the MoQToF drift cell. (I) 3D section through cell; (II) section through cell viewed from side; (III) front elevation; (IV) rear elevation. Parts are labeled as follows: (A) baratron connection; (B) gas in; (C) drift rings; (D) exit lens (L4); (E) end cap (C2); (F) cell body (C1); (G) Einzel lens (L1, L2, and L3); (H) heater terminal block; (I) mounting brackets; (J) heaters; (K) cooling line inlets; (L) feedthrough to drift rings; (M) molybdenum orifice; (N) thermocouple mounting; (O) cell screws.

baratron connection minimizes thermal transpiration effects. The drift field is created between the cell body (entrance) orifice and the end cap (exit) orifice and is kept linear by five intermediate copper guard rings connected, each separated by a precision 1 MΩ resistor. The precell Einzel lens was designed to provide the best possible focus into the drift cell. The three lens elements have the dimensions 40.6 mm o.d. × 22.4 mm i.d. × 3.8 mm. The central lens (L2) is split into four equal segments to allow x/y steering of the ions. A post cell lens (L4) was included to allow the ions to be focused out of the cell into the postcell hexapole. The cell is typically operated with 3-3.5 Torr He using 0.9 mm entrance and exit orifices with voltages of up to 65 V across the cell. All voltages prior to the drift cell, up to and including that applied to the first hexapole, float on the entrance voltage of the drift cell. The injection energy of ions into the cell is defined as the difference between the voltage on the “top hat” lens and that on the entrance to the drift cell, and it can be varied from 0 to 150 V. The instrument has been designed such that the drift cell can be removed and replaced with a short hexapole allowing the QTOF to be used (essentially) in its original configuration. Under these circumstances with a longer ion guide, we see no loss in signal intensity compared with the use of instrument prior to the modifications. Electronics. All voltages applied to the original parts of the instrument (i.e., the source, quadrupole, ToF, and ion optics) are supplied by the original instrument power supplies and controlled via Masslynx software (Waters MS Technologies Centre, Manchester, UK). Voltages applied to the cell and associated ion optics are supplied via an external power supply which was built in-house.

The bipolar pulser unit used to pulse the ions into the drift cell was also built in-house. The width of the pulse can be varied between 50 ns and 50 µs, and for all experiments described here, a width of 30 µs was used. The frequency of operation of the pulse pusher is controlled via a four-channel Stanford DG535 digital delay generator (Stanford Research Systems Inc., Sunnyvale, CA, USA) but is set to be one two-hundreth of the frequency of the pusher pulse into the ToF region of the mass spectrometer. The triggering of the ion gate pulse starts the timing of our mobility experiment, via a synchronous pulse to the QToF control electronics. Ion Mobility Measurements. For ion mobility experiments, ions are accumulated in the first hexapole by raising the potential on the hexapole top hat. Ions are then pulsed out of the hexapole using the pulser unit described above. A simultaneous pulse is sent from the pulse generator to the instrument TDC card to signal the start of an ion mobility experiment. The software then “bins” the next 200 scans of the ToF (each scan having a width equivalent of one pusher period; typically 30-85 µs) to build up a total ATD (tATD). The pusher period used is determined by the highest m/z recorded in the experiment and is always set to be as short as possible to provide the shortest bin width for ion arrival data. Data produced by the software are visualized as total ion chromatograms made up of groups of 200 ToF scans, in which is located the tATD. Extracted ion chromatograms for any ion in the spectrum, containing mass selected ATDs, can be obtained from any mass spectrum. For the data reported here, an average arrival time distribution is used for a given m/z ion and is obtained by summing over at least 10 mobility experiments. From these averaged plots of ion intensity against scan number, arrival times are calculated by multiplying the scan number by the pusher period. The arrival time, tarrival, measured for an ion is a combination of the time spent in the drift cellsthe drift time, td, and the “dead” time, tdead, spent in the ion optics between the first hexapole and the cell and in the ion optics between the cell exit and pusher. To obtain an ion’s drift time, its dead time must be accounted for. The dead time is independent of the time spent in the drift cell (and hence of the applied drift voltage), and it can be obtained by measuring ion arrival times at different drift voltages and plotting these values as a function of P/V. From eq 2, it is clear that the gradient of the line obtained from this plot is inversely proportional to the reduced mobility, K0, where L is the drift length; T is the temperature; P is the cell pressure; V is the drift voltage; and P0 and T0 are the standard pressure and temperature

tarrival ) td + tdead )

L2T0 P + tdead K0P0T V

(2)

The reduced mobility obtained from the plot is used to calculate ion cross sections using eq 3 where z is the number of charges carried by the ion; N0 is the buffer gas number density at standard pressure and temperature; µ is the reduced mass of the ion and buffer gas; and Ω is the ion’s rotationally averaged, collision cross section. K0 )

( )( ) 3ze 16N0

2π µkbT

1/2

1 Ω


(3) 6339

Table 1. Instrument Specifications before and after Modificationsa Q-ToF I Cell Chamber Pressure (no gas in cell) Cell Chamber Pressure (3.5 Torr He in cell) Analyzer Chamber Pressure (no gas) Analyzer Chamber Pressure (3.5 Torr He) ToF Chamber Pressure (no gas) ToF Chamber Pressure (3.5 Torr He) Quadrupole Mass Range Quadrupole Resolution ToF Mass Range ToF Resolution Transmission Efficiency (MS 3.5 Torr He)* Transmission Efficiency (IMS, 3.5 Torr He)* IMS Resolution** IMS Pressure Range*** IMS Temperature Range

MoQToF

n/a

1 × 10

n/a

1 × 10

6 × 10-6 mBar

1 × 10-6 mBar

n/a

1 × 10-5 mBar

-6

-3

3 × 10

-7

mBar

2 × 10

-7

mBar mBar

mBar

n/a

5 × 10

2-4500 m/z ∼500 1-5000 m/z 8000 n/a

2-32000 m/z ∼250 1-100000 m/z 8000 5%

n/a

8%

n/a n/a n/a

∼20 e4.5 Torr 100-600 K

-7

peptide [M [M [M [M [M [M

+ + + + + +

3+

3H] 4H]4+ 5H]5+ 6H]6+ 7H]7+ 8H]8+

Defr1 Y5C

Defr1 Glt

Defr1 Red

523 538 605 -

513 595 642 -

551 581 652 735

DEFB10750 627 746 811 864 938

a All values are given in Å2. The values for DEFB107 have previously been reported.50

mBar

a

*, As compared to ion transmission prior to upgrade; **, calculated for [M + 8H]8+ cytochrome C at Vd ) 60 V, P ) 3.5 T, and T ) 305 K; ***, with orifices of 0.9 mm.

Sample Preparation. Cytochrome C, ubiquitin, and lysozyme were obtained from Sigma Aldrich (Poole, Dorset, UK). Derf1 and Defr1 glt were synthesized by Albachem UK. Derf1 was reduced using DTT. Defb14 and the defensin related peptides (Dips) were made in-house using automated peptide synthesis. This was carried out on an Applied Biosystems model 433A peptide synthesizer using Rink amide AM resin for peptide amides, preloaded NovaSynTGT resin for peptide acids, and Fmoc amino acids from Novabiochem. LC-Mass spectra to confirm identity and purity were obtained on a Micromass Quattro LC mass spectrometer. Samples were prepared for nano-ESI in 50:50 methanol/water with 0.1% formic acid at concentrations ranging from 20 to 40 µM. Chemotaxis assays were performed as reported previously.55 Mobility Measurements. For all systems studied, measurements were taken with ∼3.5 Torr He in the drift cell at several drift voltages ranging from 60 to 25 V and 35 V injection voltage at ∼300 K. Pusher periods used ranged from 72 µs for cytochrome C to 60 µs for the D14ips. Molecular Modeling. Molecular modeling was performed on the β-defensin Defr1 Y5C (see Table 2 for the sequence) using software included in the AMBER 8 package.56 Model structures of Defr1 Y5C in vacuo (carrying its “physiological” net charge of +5) possessing all of the 15 possible disulfide pairings were calculated using a simulated annealing algorithm that invokes a mobile disulfide bridge methodology employing a probabilistic (55) Taylor, K.; Clarke, D. J.; McCullough, B.; Chin, W.; Seo, E.; Yang, D.; Oppenheim, J.; Uhrin, D.; Govan, J. R. W.; Campopiano, D. J.; MacMillan, D.; Barran, P. E.; Dorin, J. R. J. Biol. Chem. 2008, M709238200. (56) Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, D. A.; Pearlman, D. A.; Crowley, M.; Brozell, S.; V., T.; H., G.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; C., S.; Caldwell, J. W.; Ross, W. S.; Kollman, P. University of California: San Francisco, 2004.

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Table 2. Experimental Cross Sections of Defr1 and Related Peptidesa


bonding algorithm.57 The starting structures were each subjected to energy minimization and then to molecular dynamics at a higher temperature (600 K) followed by stepwise cooling to 0 K and energy minimization. This procedure was repeated 10 times for each topology. Mobilities and collision cross sections of the calculated conformers were estimated using the trajectory method, implemented in the Mobcal program.11 RESULTS AND DISCUSSION: General Performance of the MoQToF. The performance of the MoQToF compared to that of a QToF 1 is summarized in Table 1. The resolution was obtained from measurements on the [M + 8M]8+ cytochrome C ion. Transmission efficiency was obtained using the same concentration (20 µM) of solutions of NaCsI and the proteins Ubiquitin and Defb107. The numbers given are based on a comparison of the counts per second in the total ion chromatograms, with that obtained pre modification being set to 100%. We have constructed an instrument which is capable of obtaining both mass and mobility data. Transmission of ions is reduced due to the presence of the drift cell; none-the-less, the benefits of obtaining both mass and mobility information in a single experiment is certainly attractive. By converting our Quadrupole to act as a high m/z filter, we have also lost some resolution in this part of the instrument due to the change in the operating frequency; however, this does not affect our ToF resolution and does not impede the types of measurements we describe here. On the basis of the width of the ATDs and also on the length of our drift cell, we find that the experimental error in our recorded cross sections is on the order of 5%. In the next sections, we describe the application of the MoQToF to examine gas-phase conformations of biomolecules. Cytochrome C. Figure 3A shows ATDs for [M + 8H]8+, [M + 12H]12+, and [M+17H]17+ charge states of cytochrome C. The two higher charge state species can be seen to yield single peaks in the ATD, and the [M + 8H]8+ ATD, however, shows evidence of a shoulder indicative of a second conformation. Figure 3B shows a plot of measured cross section against charge state along with published cross sections for cytochrome C. It can be seen that there is excellent agreement between the published data and data obtained from the MoQToF, and all values agree within 6% with most in agreement within 2%. This provides a very nice example of the effect of protonation on the conformation of a gas-phase protein. As pointed out in previous work, the collision cross sections increase as a function of the number of protons which charge the protein, due to Coulombic repulsion. (57) Eastwood, H.; Barran, P. E. Manuscript in Preparation.

Figure 3. (A) Arrival time distributions for [M + 8H]8+ (dashed line), [M + 12H]12+ (dotted line), and [M + 15H]15+ (solid line) cytochrome C at 3.5 Torr, Vdrift ) 60 V. (B) Experimental cross sections of different charge states of the protein cytochrome C obtained on the MoQToF (black stars) and from the literature (open squares, Shelimov et al.14).

Figure 4. Experimental cross sections for the proteins Ubiquitin (A) and Lysozyme (B) obtained from the MoQToF (black stars) and literature (open squares; for Ubiquitin, Li et al.,40 and for Lysozyme, Valentine et al.18).

Ubiquitin and Lysozyme. Figure 4A is a plot of cross sections obtained from the protein ubiquitin measured on the MoQToF against charge state along with published cross sections. Once more, there is good agreement between the MoQToF data and previously published data (within 4%). Lysozyme is a 129 amino acid protein (∼14 kDa) which contains four intermolecular disulfide linkages. The presence of these disulfide bonds has been shown to have a stabilizing effect on the gas phase conformations adopted by the protein. Here it was studied in its fully disulfide intact (oxidized) form. Figure 4B shows a plot of cross sections measured in this work for the observed charge states ([M + 5H]5+

to [M + 10H]10+). Once more, only single resolvable conformations were observed here for each charge state. Valentine et al.18 however report the presence of a number of conformations for [M + 7H]7+ to [M + 11H]11+, and they categorize these conformations as either highly folded, partially unfolded, or unfolded (with the latter category only evident for fully disulfide reduced lysozyme). The [M + 5H]5+ and [M + 6H]6+charge states of lysozyme exist as only single conformers categorized as highly folded. The cross-sections observed in this work for the three lowest charged species ([M + 5H]5+, [M + 6H]6+, and [M+7H]7+) agree well with the values from Valentine et al. assigned as highly folded. The cross sections measured here for [M + 8H]8+, [M + 9H]9+, and [M + 10H]10+ charge states also agree well with the reported values for partially folded lysozyme. The conformational change can be seen clearly in Figure 4B. It is worth noting that Valentine et al.18 only report the partially unfolded species at high injection voltage (120 V) and not at the 30 V used here. The reason the partially unfolded conformation is sampled here is not clear, but it may be due to RF heating of the ions while they are being stored in the precell hexapole. Defensin Mobility: Conformational Preferences. Defensins are small, cationic, antimicrobial peptides that form an important part of the innate immune system of all mammals. Previous work in our group has focused on the characterization of a class of defensins known as β-defensins.50,55,58,59 These are characterized by the presence of six cysteine residues with a well-defined disulfide connectivity (Cys1-Cys5, Cys2-Cys4, Cys3-Cys6). We have examined some variations of this connectivity in naturally occurring defensins with less than six cysteines50,58 and also by synthesizing variant defensin peptides which have an altered disulfide topology. Sequences of example peptides from both of these classes are given in Figure 5, along with disulfide topologies, which have been determined using peptide mass mapping as reported previously.50,60 For comparison, we include the naturally occurring murine defensin MBD8, which is encoded by Defb8, an allele of the C57Bl/6 Defr1 gene.61 We have applied IM-MS, coupled with biological assays, to probe the structure function relationships of this important class of peptides. Table 2 shows the cross sections obtained from IM-MS measurements on the defensin peptides listed in Figure 5, along with that for the disulfide reduced form of Defr1. For the first three peptides where very small chemical changes distinguish between the sequences, the power of IM-MS to reveal conformational differences is apparent. Despite possessing seven basic residues and being electrosprayed from acidified solutions, the fully disulfide-bridged peptide Defr1 Y5C and the related peptide Defr1 Glt are found in only three low charge states, each indicative of conformationally restricted ions, with buried or partially buried amino acids. By contrast, the reduced form of this (58) Campopiano, D. J.; Clarke, D. J.; Polfer, N. C.; Barran, P. E.; Langley, R. J.; Govan, J. R. W.; Maxwell, A.; Dorin, J. R. J. Biol. Chem. 2004, 279, 48671– 48679. (59) Taylor, K.; McCullough, B.; Clarke, D. J.; Langley, R. J.; Pechenick, T.; Hill, A.; Campopiano, D. J.; Barran, P. E.; Dorin, J. R.; Govan, J. R. Antimicrob. Agents Chemother. 2007, 51, 1719–1724. (60) Campopiano, D. J.; Clarke, D. J.; Polfer, N. C.; Barran, P. E.; Langley, R. J.; Govan, J. R.; Maxwell, A.; Dorin, J. R. J. Biol. Chem. 2004, 279, 48671– 48679. (61) Morrison, G. M.; Rolfe, M.; Kilanowski, F. M.; Cross, S. H.; Dorin, J. R. Mamm. Genome 2002, 13, 445–451.


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Figure 5. Sequences and disulfide connectivities for full length defensins studied here and for Defb14 related peptides. For Defr1 Glt and for DEFB107, the symbol * refers to the presence of a Glutathionine capping group on the unpaired cysteine.

Figure 6. Use of IM-MS paired with molecular modeling to filter possible disulfide connectivities of synthetic defensins. The shaded area represents the experimental measurement for the [M + 5H]5+ ion of 605 Å2 along with the expected error of 5%. The squares represent the collision cross sections calculated using MOBCAL11 from minimized structures of the defensin Defr1 Y5C, obtained using the AMBER force field.71 The 15 possible arrangements for disulfide bridging involving all of the six cysteines are shown. (A) 1-2, 4-5, 3-6; (B) 1-2, 4-3, 5-6; (C) 1-2, 4-6, 5-3; (D) 1-3, 2-4. 5-6; (E) 1-3, 2-5, 4-6; (F) 1-3, 2-6, 4-5; (G) 1-6, 2-3, 4-5; (H) 1-6,2-3, 4-5; (I) 1-5, 2-3, 4-6; (J) 1-5, 2-4, 3-6; (K) 1-5, 2-6, 3-4; (L) 1-6, 2-4, 3-5; (M) 1-4, 2-3, 5-6; (N) 1-4, 2-6, 3-5. J is the canonical fold.

peptide also shows a [M + 6H]6+ ion, and the least restricted disulfide-bridged peptide DEFB107 presents a wider charge state distribution. The cross sectional measurements are also enlightening. For DEFB107 and the reduced form of Defr1, the collision cross sections increase with charge state in a similar fashion to that observed for Ubiquitin and cytochrome C above. The more restricted peptides Defr1 Y5C and Defr1 Glt show a smaller increase in collision cross section as a function of charge state, which shall be discussed in more detail in the next section. Use of IM-MS to Predict Disulfide Pairing. As can be seen from the data presented above and in previous work,13,18,62,63 (62) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1997, 8, 954–961. (63) Jarrold, M. F. Acc. Chem. Res. 1999, 32, 360–367.

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IM-MS can be used to reveal the extent of protein unfolding as a function of charge state. As the number of protons increases, so does the collision cross section, as Coulombic repulsion drives apart noncovalently bonded regions. For a protein which is structurally restrained, for example, by disulfide bridges, this increase in collision cross section will be limited by covalent linkages. This is illustrated in Figure 4B above for Lysozyme, where after a partial unfolding between [M + 7H]7+ and [M + 9H]9+ the extent of “unfolding” appears to decrease. This is attributable to restriction from the presence of two disulfide bridges.18 For defensins and many other proteins which have large numbers of disulfide bridges, it is often difficult to correctly assign which cysteines are paired together. The favored approach is to use a combination of enzymatic digestion peptide mass mapping with mass spectrometry and/or Edman degradation, along with partial selective reduction-alkylation of cysteines.64–66 These methods are experimentally challenging and often very sample demanding. For synthetic and recombinant peptides and proteins possessing large numbers of cysteines, it is likely that they will be produced with a mixture of disulfide pairings, which can cause difficulties with both structural and functional assays, as well as limit the validity of such approaches to generate therapeutic agents.67 Some recent work has sought to use top-down sequencing as a method to determine disulfide bridging, and this certainly has promise.68 We present here the use of IM-MS combined with molecular modeling to reduce the search space for possible disulfide pairings. It is clear from the data shown in Table 2 that the reduced Defr1 is able to form considerably larger ions than Defr1 Y5C. The less disulfide restricted DEFB107 is also able to unfold more easily. To see if the limited conformation space for the Defr1 Y5C ions can be related to the known disulfide pairings, we calculated low-energy structures for a [M + 5H]5+ ion based on the physiological charge state of the peptide for all 15 of the possible arrangements of disulfide bridges between the six cystine residues. For each disulfide topology, the collision cross section of ten low-energy structures was obtained (64) Merewether, L. A.; Le, J.; Jones, M. D.; Lee, R.; Shimamoto, G.; Lu, H. S. Arch. Biochem. Biophys. 2000, 375, 101–110. (65) Watson, J. T.; Yang, Y.; Wu, J. J. Mol. Graph Model 2001, 19, 119–128. (66) Zhou, Z.; Smith, D. L. J. Protein Chem. 1990, 9, 523–532. (67) Walsh, G. Trends Biotechnol. 2005, 23, 553–558. (68) Zhang, M.; Kaltashov, I. A. Anal. Chem. 2006, 78, 4820–4829.

Table 3. Experimental Collision Cross Sections of Defb14 and Related Peptidesa peptide 55

chemoattracts [M + 2H]2+ [M + 3H]3+ [M + 4H]4+ [M + 5H]5+ [M + 6H]6+ [M + 7H]7+ [M + 8H]8+ a

Defb14

Defb14 1CV

Defb14 0C

Defb14 Dip1

Defb14 Dip2

Defb14 Dip3

709 751 881 1008 -

916 962 1018 1105

× 979 1070 1170

× 465 546 -

× 444 486 585 -

× 434 491 580 -

All values are given in Å2.

and averaged. Figure 6 summarizes our findings. The experimental value of 605 Å2 is lower than that obtained for the canonical fold but still lies within experimental error. Of the 15 possible connectivites, 10 lie well outside the experimental region, so we have indeed filtered the possible disulfide bridging arrangement. Of course, we have not properly surveyed the folding surface of these peptides by only calculating 10 structures, but we wished to demonstrate here a method that could be rapidly applied. It is clear that topologies with more open parings (1-2, 3-4, 5-6 being an obvious example) will form more extended gas-phase conformations, and those more like the canonical will be more restricted. Because of this, the filtering described would be feasible by a combination of experimental data and results from more coarse-grained modeling. If IM-MS was performed on a protein with a mixture of topologies, these would be revealed by a significant broadening of the ATD for a given ion. For proteins with several cystine residues, such an approach could be extremely useful in determining whether a given protein had formed one or more disulfide-bridged folds, and this in turn would give insight to folding pathways. Insights to Chemotactic Properties from Ion Mobility Data. As well as their antimicrobial activities, defensins also act as powerful chemoattractants.69 Recently, we have synthesized a set of novel peptides based around the β-defensin Defb14,55 the murine orthologue of human β-defensin 3. The peptides described are: full length Defb14 with three disulfide bonds; Defb14 1Cysv, a full length peptide with all but the penultimate cysteine mutated to alanine; Defb14 0C, a full length peptide with all cysteines mutated to alanine; Dip1, the N-terminal half of Defb14 Ala; Dip2, the C-terminal half of Defb14 Ala; and Dip3, the C-terminal half of Defb14 1Cys. This group of defensin and defensin inspired peptides (D14ips) makes an ideal test set for the MoQToF. Figure 5 shows the sequences of these six peptides. Table 3 shows the measured cross sections for all observed charge states of each of these peptides, along with details on whether or not each peptide chemoattracts CCR6-transfected human embryonic kidney (HEK)293 cells.55 Figure 7 shows the change in cross section with charge state for all of the full length peptides. The disulfide intact form (Defb14) can be seen to present a more compact form than the two other peptides, especially at lower charge. (69) Yang, D.; Biragyn, A.; Kwak, L. W.; Oppenheim, J. J. Trends Immunol. 2002, 23, 291–296. (70) Li, J. W.; Taraszka, J. A.; Counterman, A. E.; Clemmer, D. E. Int. J. Mass Spectrom. 1999, 187, 37–47. (71) Case, D. A.; Cheatham, T. E., 3rd; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M., Jr.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. J. Comput. Chem. 2005, 26, 1668–1688.

Figure 7. Experimental cross sections of Defb14 (squares), Defb14 1Cv (circles), and Defb14 0C (triangles). The dotted line represents the average cross section of the 20 NMR structures of the human equivalent of Defb14, HBD-3,72 which is found to be 883 Å2 using MOBCAL.11

The data at low charge states fit well with those from the NMR structure of the Human ortholog of Defb14, HBD3. This is a clear indication again that disulfide bonds conformationally restrict the peptide: for example, for the [M + 5H]5+ ion, Defb14 (which has three disulfide bonds) is ∼18% smaller than Defb14-1Cv (0 disulfides). Comparison of Defb14-0c and Defb14-1Cysv shows a surprising influence of the substitution of a cysteine for an alanine (essentially the substitution of an -SH group for an -H). Given the minimal nature of the substitution, one might expect the two peptides to behave in much the same manner. The data shown here, however, shows Defb14-0c to present a more extended conformation than Defb14 1CV at all charge states studied; clearly, the cysteine residue confers a more compact structure on the peptide. This different behavior is mirrored in vitro where experiments have shown Defb14 1CV to be an active chemoattractant while Defb14-0c is not (Table 3 and in Taylor et al.55). It is tempting to speculate that the more compact gas-phase geometry presented in the 1CysV form of Defb14 is also present in solution allowing the correct conformation to be adopted for receptor binding and directional cell movement to occur. The IM-MS data from the Defb14 Dips is also insightful (Table 3). The C-terminal portion of the peptide (Dips 2 and 3) presents a more compact gas-phase structure than the N-terminal half (Dip1). Interestingly, despite having the same alanine to (72) Schibli, D. J.; Hunter, H. N.; Aseyev, V.; Starner, T. D.; Wiencek, J. M.; McCray, P. B., Jr.; Tack, B. F.; Vogel, H. J. J. Biol. Chem. 2002, 277, 8279– 8289.


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cysteine change as Defb14-0c or Defb14-1CV, Dip2 and Dip3 have virtually identical cross sections at all charge states studied. This suggests that it is not simply a cysteine to alanine change which confers the more compact geometries observed for Defb14 1CV but rather an intramolecular interaction between the cysteine and the N-terminal half of the peptide. If we again examine the situation in vivo, we find neither Dip2 nor Dip3 is chemotactically active. In other words, chemotactic activity is dependent on the presence of both the N-terminus and a C-terminal cysteine. This is also the requirement for more compact gas-phase geometry. SUMMARY A new ion mobility mass spectrometer, the MoQToF, has been presented. The instrument has been shown to produce reliable ion mobility data in good agreement with previously published data. It is capable of producing mobility measurements over a wide temperature range. The coupling of a high m/z capable QTOF which possesses an ion source well suited to native electrospray ionization for proteins means that this IM-MS instrument could have a role in obtaining accurate cross-sections of large native proteins and their complexes. We have presented the use of IM-MS measurements coupled with molecular modeling to filter the possible disulfide connectivities for cysteine-rich proteins. Our ion mobility studies of Defr1, Defb14, and related peptides have revealed the influence of

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disulfide bonding on gas-phase geometry and in particular the influence of intramolecular interactions on the geometries adopted by the peptides. We also present a qualitative link between the extent of unfolding of linear forms of Defb14 and chemotatic properties, which suggest that the presence of a single cysteine in position 5 is sufficient to confer structural stability to this defensin, but when substituted for an alanine, this stability is lost. These insights from gas-phase studies have important implications for a molecular understanding of how changes in sequence and disulfide bridging influence the activity of defensin peptides. ACKNOWLEDGMENT This research was supported by the EPSRC grants GR/ S77639/01 and EP/C541561/1 and in particular via the award of an Advanced Research Fellowship to P.E.B. and studentships to H.E. and B.J.M. We also had support from the Royal Society, the British Mass Spectrometry Society, and Waters MS Technologies Centre, and in particular, we thank Steven Pringle, Kevin Giles, Jason Wildgoose, and Robert Bateman. We also are grateful for the continuing support from the School of Chemistry at the University of Edinburgh. Received for review April 1, 2008. Accepted June 10, 2008. AC800651B

Development of an Ion Mobility Quadrupole Time of Flight Mass

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