Ion mobility spectrometry (IM) has become an

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1 Journal of Biomolecular Techniques 3: ABRF R AB F REVIEWS A Fundamental Introduction to Ion Mobility Mass Spectrometry Applied to the Analysis of Biomolecules Guido F. Verbeck, Brandon T. Ruotolo, Holly A. Sawyer, Kent J. Gillig, and David H. Russell The Laboratory for Biological Mass Spectrometry, Texas A&M University, College Station, Texas Chemists are constantly striving for techniques that add dimensions of orthogonality with increased throughput and sample complexity. Ion mobility spectrometry (IM) is a gasphase separation method that adds new dimensions to mass spectrometry (MS). IM separates gas-phase ions based on their collision cross-section and can be coupled with timeof-flight (TOF) mass spectrometry to yield a powerful tool used in the identification and characterization of proteins and peptides.a fundamental introduction to IM is presented with a focus on resolution, sensitivity, and orthogonality coupled with TOF-MS. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: David H. Russell ( russell@mail.chem.tamu.edu). Ion mobility spectrometry (IM) has become an important analytical tool over the past 5 years. Known also as plasma chromatography and ion chromatography, it has mainly been applied to the analysis of volatile organic compounds,3 and used as a tool to probe the electronic states of ions. 4 Recently, ion mobility has been applied to the analysis of biomolecules employing electrospray ionization (ESI) 5 8 and matrix-assisted laser-desorption ionization (MALDI) sources. 9 These combined with mass spectrometry create a powerful tool in the analysis of proteins and peptides. Ion mobility separates ions based on their mobility through a neutral target gas, usually helium. This separation is achieved by the migration of the ions in a linear field, E, through the buffer gas at a specific pressure, p. This is normally expressed as the combined term E/p. Figure shows a basic diagram of a MALDI-IM-oTOF instrument used in our lab. Once the ions have eluted from the mobility cell, they are pulsed orthogonally into the time-of-flight (TOF) chamber and detected. Ion mobility adds a degree of orthogonality to mass spectrometry due to the ion s interaction with a neutral target or buffer gas. This interaction for macromolecules leads to separation based primarily on the ion s volume. Ions having similar conformational forms will display a trend line with respect to massto-charge, making it easy to isolate peptides and proteins with conformational differences as outliers of these trend lines. In this paper a fundamental introduction to ion mobility as it applies to macromolecules, specifically biomolecules, is presented. Current trends in analytical nomenclature and calculations from groups active in this field are also reviewed. The analysis of D IM- MS data is illustrated by an IM-TOF spectrum of a tryptic digest of bovine hemoglobin. 56 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 3, ISSUE, JUNE 00

2 ION MOBILITY MASS SPECTROMETRY TOF Detector N Laser TOF Region Concentric Ring System Mobility Detector Direct Insertion Probe Ion Mobility Cell Orthogonal Exraction FIGURE Instrument schematic of a MALDI IM orthogonal TOF mass spectrometer. ION MOBILITY Ion mobility spectrometry separates ions based on their different drift velocities ( d ) through a buffer gas at an applied electric field (E). d KE [] The mobility (K) is the constant that defines this ion s drift. This proportionality constant is usually reported as reduced mobility (K 0 ), the ion s mobility at standard temperature and pressure. p 73.5 K 0 K [] 760 T The mobility of an ion through a buffer gas is dependent on several factors such as the ion charge (q), the number density of buffer gas (N), the reduced mass of the ion-neutral complex (µ), the absolute gas temperature (T), and the ion s collision cross-section ( 0 ). 3 q K 6 N k b T [3] We see here in the fundamental ion mobility equation that the mobility is inversely proportional to the square of the reduced mass and the ion s collision cross-section. In the analysis of macromolecules, the value µ approaches the mass of the buffer gas, and thus can be 0 treated as a constant for ions with mass greater than 0.5 kda. Therefore, the mobility term becomes dependent only on the collision cross-section of the ion. 3 K [4] 0 For these macromolecules, o encompasses the scattering angle between the ion and the buffer gas, the relative velocities (v rel ) of each, and the orientationally averaged geometry (b) of the ion. 4 This further simplifies the above expression by putting the mobility in terms of predictable variables. K [5] bv rel Since the reduced mass for large molecules is approximately constant, the orientationally averaged collision cross-section plays the major role in macromolecular ion separation in ion mobility. 5 IM-MS DATA ANALYSIS When developing a multidimensional technique, it is desirable that the dimensions evaluate unrelated molecular properties of the analyte of interest. When the dimensions of a technique are completely JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 3, ISSUE, JUNE 00 57

3 G. F.VERBECK ET AL. independent of each other they are said to be orthogonal. 6 If the molecular properties analyzed by the dimensions are not completely unrelated, a multidimensional technique can be reduced to a one-dimensional technique with signal distributed along a diagonal. As can be seen from the massmobility plot of C 60 /C 70 derived from carbon clusters, shown Figure A, the signal is distributed along a trend line, indicating that the dimensions of IM-MS have a large amount of cross-information, i.e., there is a high degree of correlation between the mobility and mass of an ion. The large amount of cross-information present in a mass-mobility plot does not reduce the utility of IM- MS; rather the cross-information guides the analysis of the complex mixture. 7 For example, the analysis of complex mixtures is often directed by the presence of multiple trend lines in a mass-mobility data set, as illustrated by Figure B, which combines trend lines for both peptides and carbon cluster fragment ions. Trend lines are present in the mass-mobility plots due to the high correlation between an ion s mass and collision cross-section. Addition of mass within an ion series can be likened to adding subunits to a polymer; as the subunits are added, the gas-phase volume is increased in a linear fashion over a limited mass range. C 60 and related carbon clusters have a very tight and uniform (spherical) conformation, resulting in a highly linear mass-mobility trend, whereas peptide ions have a more open conformation. Due to the differences in which different ion classes increase their collision cross-section upon mass addition, different trend lines result. A B FGURE MALDI-IM-TOF mass spectra of (A) carbon cluster fragments from 90% C 60 and 0% C 70 and (B) the peptide substance P with carbon cluster fragments. 58 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 3, ISSUE, JUNE 00

4 ION MOBILITY MASS SPECTROMETRY The trend lines consisting of peptide ions sometimes contain significant deviations from the expected linear relationship, indicating that the correlation between the ion s mass and collision cross-section is reduced. These deviations are highly visible due to the usually high amount of cross-information in a massmobility plot. Ions that have deviations from the trend line have a structure that is radically different from that of the other ions in the series. An example of such a deviation is seen in the mobility plot of bovine hemoglobin digest fragments shown in Figure 3. The two tryptic digest fragments of bovine hemoglobin elute in reverse mass order because of a distinct volume difference. The 75-m/z peptide is proposed to have a more compact structure opposed to a more extended helical 66-m/z peptide. This mobility profile is distinctly different from those usually observed, as demonstrated by the inset of Figure 3, which shows a mobility profile of substance P and -melanocyte stimulating hormone. The fine structure exhibited by deviations from a trend line can be analyzed with collision cross-section calculations and fitting of mobility peak profiles as well as with computational and other experimental techniques such H/D exchange. RESOLUTION Traditional mass spectrometry bases resolution on the mass; however, since resolution in ion mobility is more complex, it has become common practice to compare IM resolution with chromatography resolution. This practice, set in motion by Hill and co-workers, 8 0 can be accomplished by starting with the definition of resolution (R) for an arrival time plot, assuming a gaussian shape. t t R [6] t W Because there is a difference between chromatographic and IM peak shapes, the base width, W, is 4.7 times the standard deviation ( ) for the IM arrival time peak instead of 4 as in chromatography. The peak width is defined as the sum of four peak broadening terms: the initial pulse width, ion diffusion through a target gas, space/charge effects, and ion/target gas interaction. W W i W d W s W a [7] FIGURE 3 MALDI-IM plot of two tryptic digest peptides LLGNVLVVVLAR, m/z 66, and LLLVVYPWTQR, m/z 75, of bovine hemoglobin. Inset: MALDI-IM plot of substance P (m/z 347) and -melanocyte stimulating hormone (m/z 665). JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 3, ISSUE, JUNE 00 59

5 G. F.VERBECK ET AL. Experiments are usually designed to limit the number of band-broadening terms. In the case of macromolecules with small initial pulse times, Equation 7 can be reduced to include only the diffusion-broadening term. The spatial standard deviation ( ) of diffusion is defined as Dt d [8] where D is the ion s diffusion coefficient and t d is time in the drift chamber. Diffusion perpendicular to the applied field is defined by the Nernst Einstein relation, k b T D K [9] q where K is the ion s mobility at an absolute temperature T. The diffusion width can now be defined as W d 4.7 [0] in terms of the drift velocity (v d ). Again, assuming that the diffusion width is much greater than the other three peak broadening terms, then W W d and the ion mobility peak resolution is qel 44.k b T 44.k b Tv d t d R [] Here, some fundamental concepts can be visualized. Resolution is a function of applied electric field and drift length and inversely proportional to absolute temperature. SENSITIVITY Ion detection with ion mobility is performed primarily with sensitive detectors (i.e., microchannel plate detectors) because of ion loss due to diffusion within the mobility cell. This, for example, is at least an order of magnitude greater than that of TOF detection. The attenuation of ions through the exiting orifice is dependent on the radius of the orifice (r c ), the diffusion of the ions perpendicular to the field (D ), and the time to transverse the mobility cell (t). r c qe A exp 4D [] t d At low applied fields on the mobility cell, the diffusion can be easily calculated using the Nernst Einstein relation (Eq. 9); however, when the applied field is increased, the field itself becomes a concern. The ion no longer transfers all its translational energy into the target gas. The Wannier equation modifies Einstein s relation to include the field strength term., kt q 3 m M m.908m D K m [3] A C 60 cluster ion in He buffer gas at room temperature and Torr with an applied field of 0 V/cm will have ~3% transmission through a 0.5-mm orifice. If the voltage is increased to 00 V/cm, then the transmission is only increased to ~4%. One of the major concerns with ion mobility is the issue of sensitivity. The high field increases the resolving power of ion separation; however, this increased field has little or a negative effect on ion transmission. Our lab is currently developing a periodic focusing ion mobility instrument to reduce perpendicular diffusion and increase sensitivity at high applied fields. 3 CONCLUSION E K 3 q Ion mobility-mass spectrometry is becoming a powerful tool in the analysis of macromolecules, specifically proteins and peptides. Both ESI and MALDI combined with IM provide powerful ion sources for macromolecular analysis. It is becoming necessary to develop the tools to interpret the D trace and gather valuable conformational information. IM combines the degree of orthogonality with high-throughput analysis needed in the biomolecular community. REFERENCES. Karasek FW. Plasma chromatography. Anal Chem 974; 46:70A 70A.. Carr TW. Plasma Chromatography. New York: Plenum Press, 984: Eiceman GA, Karapas Z. Ion Mobility Spectrometry. Boca Raton: CRC Press, 994: Kemper PR, Bowers MT. Electronic-state chromatography: application to first-row transition-metal ions. J Phys Chem 99;95: Clemmer DE, Jarrold MF. Ion mobility measurements and their applications to cluster and biomolecules. J Mass Spectrosc 997;3: Couterman AE, Clemmer DE. Large anhydrous polyalanine ions: evidence for extended helices and onset of a more compact state. J Am Chem Soc 00; 3: JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 3, ISSUE, JUNE 00

6 ION MOBILITY MASS SPECTROMETRY 7. Wu C, Siems WF, Klasmeier J, Hill HH. Separation of isomeric peptides using electrospray ionization/highresolution ion mobility spectrometry. Anal Chem 000; 7: Wyttenbach T, Kemper PR, Bowers MT. Design of a new electrospray ion mobility mass spectrometer. Int J Mass Spect 00;: Koomen JM, Ruotolo BT, Gillig KJ, McLean JA, Kang M, Fuhrer K, Gonin M, Schultz JA, Dunbar KR, Russell DH. Oligonucleotide analysis with MALDI-ion mobility- TOF MS. Submitted to J Anal Bioanal Chem. 0. Ruotolo BT, Verbeck GF, Thomson LM, Woods AS, Gillig KJ, Russell DH. Distinguishing between phosphorylated and non-phosphorylated peptides with ion mobility-mass spectrometry. Submitted to J Proteomics Res.. Stone EG, Gillig KJ, Ruotolo BT, Russell DH. Optimization of a matrix-assisted laser desorption ionization-ion mobility-surface induced dissociation-orthogonal-time-of-flight mass spectrometer: Simultaneous Acquisition of Multiple correlated MSand MS Spectra. Int J Mass Spect 00;: Mason EA, McDaniel EW. Transport Properties of Ions in Gases. New York: John Wiley & Sons, 988: Tammet H. Size and mobility of nanometer particles, clusters and ions. J Aerosol Sci 995;6: Shvartsburg AA, Jarrold MF. An exact hard-spheres scattering model for the mobilities of polyatomic ions. Chem Phys Lett 996;6: Mesleh MF, Hunter JM, et al. Structural information from ion mobility measurements: effects of the longrange potential. J Phys Chem 996;00: Asbury GR, Hill HH. Using different drift gases to change separation factors ( ) in ion mobility spectrometry. Anal Chem 000;7: Giddings JC. Two-dimensional separations: concept and promise. Anal Chem 984;56:58A 70A. 8. Ruotolo BT, Verbeck GF, Thomson LM, Gillig KJ, Russell DH. Observation of conserved solution-phase secondary structure in gas-phase tryptic peptides. J Am chem Soc Accepted. 9. Rokushika S, Hatano H, Baim MA, Hill HH. Resolution measurements for ion mobility spectrometry. Anal Chem 985;57: Asbury GR, Hill HH. Evaluation of ultrahigh resolution ion mobility spectrometry as an analytical separation device in chromatographic terms. J Microcolumn Sep 000; : Kemper PR, Bowers MT. A hybrid double-focusing mass spectrometer-high-pressure drift reaction cell to study thermal energy reactions of mass-selected ions. J Am Soc Mass Spectrom 990;: McDaniel EW, Moseley JT. Tests of the Wannier expressions for diffusion coefficients of gaseous ions in electric fields. Phys Rev A 97;3: Gillig KJ, Russell DH. A periodic field focusing ion mobility spectrometer. PCT Int Appl 00; 36pp: WO JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 3, ISSUE, JUNE 00 6