Performance Evaluation of a Hybrid Linear Ion Trap/Orbitrap Mass Spectrometer

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1 Anal. Chem. 2006, 78, Accelerated Articles Performance Evaluation of a Hybrid Linear Ion Trap/Orbitrap Mass Spectrometer Alexander Makarov,* Eduard Denisov, Alexander Kholomeev, Wilko Balschun, Oliver Lange, Kerstin Strupat, and Stevan Horning Thermo Electron (Bremen) GmbH, Hanna-Kunath-Strasse 11, Bremen Germany Design and performance of a novel hybrid mass spectrometer is described. It couples a linear ion trap mass spectrometer to an orbitrap mass analyzer via an rf-only trapping quadrupole with a curved axis. The latter injects pulsed ion beams into a rapidly changing electric field in the orbitrap wherein they are trapped at high kinetic energies around an inner electrode. Image current detection is subsequently performed after a stable electrostatic field is achieved. Fourier transformation of the acquired transient allows wide mass range detection with high resolving power, mass accuracy, and dynamic range. The entire instrument operates in LC/MS mode (1 spectrum/ s) with nominal mass resolving power of and uses automatic gain control to provide high-accuracy mass measurements, within 2 ppm using internal standards and within 5 ppm with external calibration. The maximum resolving power exceeds (fwhm). Rapid, automated data-dependent capabilities enable real-time acquisition of up to three high-mass accuracy MS/MS spectra per second. Increasing speed of chromatographic separation and complexity of analyzed mixtures provides a continuous impetus for the development of faster and simultaneously more intelligent and robust mass spectrometric detectors. Reliable separation and reliable identification of complex mixtures with multiple coeluting compounds necessarily require higher resolving power, while structural analysis by MS/MS and accurate mass determination become complementary confirmation tools. * Corresponding author. Tel: +49(0) Fax: +49(0) alexander.makarov@thermo.com. A typical example of such a mass spectrometer is the quadrupole/time-of-flight (TOF) hybrid, 1,2 wherein precursor ions are selected by a quadrupole mass filter and accurate mass determination (including analysis of fragment ions) is carried out in an orthogonal-acceleration TOF. Though very successful for a range of applications, such hybrids nevertheless suffer from low ion transmission (resulting in poor MS/MS sensitivity and detection limits) and a limited intensity range over which accurate mass data can be acquired. These shortcomings were overcome in a next-generation hybrid instrument, a linear ion trap/fourier transform ion cyclotron resonance (FTICR) mass spectrometer. 3 Like earlier FTICR hybrids, 4,5 this mass spectrometer accumulates ions externally to a superconducting magnet, but in addition, it combines high trapping capacity, MS n capabilities, and automatic gain control (AGC) of linear ion trap mass spectrometers 6 with the unsurpassed mass accuracy, dynamic range, and resolving power of FTICR mass spectrometers. The unprecedented specificity and quality of data from this combination necessitates the added complexity of the superconducting magnet. This inspired the quest for a magnetic field-free analyzer of comparable performance, which would be more compatible with the capacities of a typical laboratory. (1) Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A. J. Mass Spectrom. 2001, 36, (2) Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1996, 10, (3) Syka, J. E. P.; Marto, J. A.; Bai, D. L.; Horning, S.; Senko, M. W.; Schwartz, J. C.; Ueberheide, B.; Garcia, B.; Busby, S.; Muratore, T.; Shabanowitz, J.; Hunt, D. F. J. Proteome Res. 2004, 3, (4) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, (5) Belov, M. E.; Nikolaev, E. N.; Anderson, G. A.; Udseth, H. R.; Conrads, T. P.; Veenstra, T. D.; Masselon, C. D.; Gorshkov, M. V.; Smith, R. D. Anal. Chem. 2001, 73, (6) Schwartz, J. C.; Senko, M. W.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2002, 13, /ac CCC: $ American Chemical Society Analytical Chemistry, Vol. 78, No. 7, April 1, Published on Web 02/18/2006

2 From its first appearance as a proof-of-principle device, 7 the orbitrap mass analyzer was considered as a potential alternative for FTICR because of its high resolution and good mass accuracy. Using the principle of orbital trapping in electrostatic fields, 8 the orbitrap consists of an inner and an outer electrode, which are shaped to create a quadro-logarithmic electrostatic potential. Ions rotate about the inner electrode and oscillate harmonically along its axis (the z-direction) with a frequency characteristic of their m/z values. An image current transient of these oscillations is converted to a frequency spectrum using a Fourier transform similar to the approach used in FTICR. For a given spread of initial parameters of ions, the orbitrap provides the best performance within the smallest dimensions, relative to other types of electrostatic traps, 8 thus making it the most suitable candidate for image current detection and Fourier transform mass spectrometry. Initially, high resolving power, internal mass accuracy, and high space charge capacity were demonstrated with the orbitrap mass analyzer using pulsed ion sources. Because of high ion velocities and the absence of any collisional cooling inside the orbitrap, injection must occur in a short burst, on a time scale well below 1 ms (actually, only a few microseconds if coherence of ion packets is to be provided by the injection, thereby eliminating the need for additional excitation 9 ). For continuous ion sources, this is possible only when additional ion storage is introduced to store ions and then inject them rapidly into the orbitrap. Such implementation for injection of electrosprayed ions into the orbitrap was initially based on axial ejection of ions from a linear rf-only quadrupole As the space charge capacity of such a trap is compromised by the need to extract ions within hundreds of nanoseconds, use of large ion numbers resulted in great variation of ion kinetic energies, together with angular and spatial spreads that limited mass range, transmission, dynamic range, and mass accuracy over a wide mass range. This work involves a new approach to ion storage that is based on orthogonal, rather than axial, ion ejection from an rf-only quadrupole. 11 This implementation alleviates the above problems and paves the way for a hybrid mass spectrometer combining the tandem mass spectrometry capability of the linear ion trap mass spectrometer with the high resolution and mass accuracy capability of the orbitrap. This combination allows high-quality accurate mass MS n spectra to be acquired using brief ion accumulation periods and relatively small ion populations. Here we characterize the analytical parameters and features of this hybrid instrument. EXPERIMENTAL SECTION Figure 1 presents a schematic diagram of the LTQ orbitrap hybrid mass spectrometer (Thermo Electron, Bremen, Germany). The design, operation, and control of the front-end LTQ mass spectrometer has been described elsewhere. 6 Briefly, ions from the electrospray ion source are admitted via rf-only multipoles into the linear trap of the LTQ, wherein ions are analyzed and radially ejected to a pair of secondary electron multipliers. 6 An important feature of the instrument is the procedure of AGC, 12 wherein a short prescan is used to determine the ion current within the mass range of interest, hence enabling storage of a defined number of ions ( AGC target value ) in the subsequent analytical scan. As in the LTQ FT mass spectrometer, all modes of LTQ operation remain available, but they are complemented by the ability to analyze ions in an additional mass detector (FTICR or, as in this work, the orbitrap). In the LTQ orbitrap, a transfer octapole (300 mm long, 400 V p-p rf, 5.7 mm inscribed diameter) delivers ions into a curved rf-only quadrupole whose central axis follows a C-shaped arc (hence the name C-trap). The C-trap uses rods with hyperbolic surfaces and is enclosed by two flat lenses with apertures for ion transport through them. The plate between the octapole and C-trap forms the gate electrode and the other plate forms the trap electrode. The enclosed volume is filled with nitrogen bath gas at 1 mtorr via an open-split interface connected to the nitrogen line of electrospray ion source. Nitrogen has been chosen as a bath gas in favor of helium due to better collisional damping and lower gas carryover toward the orbitrap. The C-trap assembly is pumped by the split-flow turbopump of the LTQ mass spectrometer, as indicated in Figure 1. Ions start from the back section of the linear trap held at a dc offset of 6-10 V (here and below voltages are given for positive ions). After acceleration into the transfer octapole by a 2-10 V potential difference, ions are transferred into the C-trap held at a dc offset of 0 V with an applied rf voltage of V p-p and are reflected by the trap electrode (12-15 V). The gate electrode is always kept at a positive offset of 3-6 V. On entry into the C-trap, ions lose energy in collisions with nitrogen bath gas, these collisions being mild enough to avoid any fragmentation. Due to the relatively low gas pressure and short length of the C-trap, ions need more than one pass through the entire system to be trapped. Nevertheless, ions finally come to rest at the place where gas collisions occur with the lowest dc offset along their path, i.e., in the C-trap. As the result of collisional cooling, the ions form a thin, long thread along the curved axis of the C-trap. This thread is compressed axially by applying 200 V to both the gate and the trap electrodes. After that, the rf voltage on the quadrupolar electrodes of the C-trap is rapidly ramped down (over ns) and dc pulses are applied to the electrodes as follows: 1200 V to the push-out electrode (i.e., the electrode furthest from the center of C-trap curvature), 1000 V to the pull-out electrode (the electrode closest to the center of curvature), and 1100 V to both the upper and lower electrodes. This voltage distribution forces ions orthogonally to the axis of the C-trap (center of curvature of the C-trap) where they leave via a slot in the pull-out electrode. Unlike axial ejection, 9,10 fast and uniform extraction is provided for large ion populations. After leaving the C-trap, the ions pass through appropriately curved ion optics, are accelerated to high kinetic energies, and converge into a tight cloud, which is able to pass through a small entrance aperture and enter the orbitrap tangentially. On their way from the C-trap, ions pass through three stages of differential pumping until they reach the ultrahigh vacuum compartment of (7) Makarov, A. A., Anal. Chem. 2000, 72, (8) Kingdon, K. H. Phys. Rev. 1923, 21, (9) Hardman, M. E.; Makarov, A. A. Anal. Chem. 2003, 75, (10) Hu, Q.; Noll, R.; Li, H.; Makarov, A. A.; Hardman, M. E.; Cooks, R. G. J. Mass Spectrom. 2005, 40, (11) Makarov, A. A.; Denisov, E.; Lange, O.; Kholomeev, A.; Horning, S. Proc. 53rd Conf. Am. Soc. Mass Spectrom., San Antonio, TX, June 5-9, 2005; Poster (12) Schwartz, J. C.; Zhou, X. G.; Bier, M. E. U.S. Patent 5,572, Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

3 Figure 1. (A) Schematic layout of the LTQ orbitrap mass spectrometer: (a) Transfer octapole; (b) curved rf-only quadrupole (C-trap); (c) gate electrode; (d) trap electrode; (e) ion optics; (f) inner orbitrap electrode; (g) outer orbitrap electrodes. (B) Simplest operation sequence of the LTQ orbitrap mass spectrometer (not shown are the following: optional additional injection of internal calibrant; additional MS or MS n scans of linear trap during the orbitrap detection) the orbitrap sustained at mbar. The ion optics assembly is pumped by two 70 L/s TMH-71P turbopumps and the orbitrap by a 250 L/s TMU-262 turbopump (Pfeiffer Vakuum GmbH, Asslar, Germany). To avoid direct carryover of gas from the C-trap to the orbitrap, ions are displaced in the vertical direction using a dual electrostatic deflector. 9 The short transfer distance reduces deleterious time-of-flight separation and thus minimizes differences in intensity distributions between mass spectra acquired with the linear trap and orbitrap mass analyzers. Ions are captured in the orbitrap by rapidly increasing the electric field 7,9,10 and gradually spread into rotating thin rings oscillating axially along the inner electrode. The inner diameter of the orbitrap outer electrodes is 30 mm, and the maximum outer diameter of the inner electrode is 12 mm. Axial oscillations are initiated by injecting ions at an offset of 7.5 mm relative to the equator of the orbitrap, eliminating the necessity of any excitation. Ion packets are injected into the orbitrap with a duration much shorter than one axial oscillation, ensuring coherent motion. A deflector electrode near the entrance slit of the orbitrap is used to compensate fringing fields. Detection of image current from coherent ion packets takes place after the voltage on the inner electrode has been stabilized at 3.5 kv. Signals from each of the outer electrodes are amplified by a differential amplifier and transformed into a frequency spectrum by fast Fourier transformation. These frequencies relate to axial oscillations of ions along the orbitrap, which are independent of the energy and spatial spread of the ions. The typical frequency for m/z ) 524 is 300 khz. Single zero-filling and Kaiser-Bessel apodization 13 is used to improve the peak shape. The frequency spectrum is converted into a mass spectrum using a two-point calibration and processed (13) Goodner, K. L.; Milgram, K. E.; Williams, K. R.; Watson, C. H.; Eyler, J. R. J. Am. Soc. Mass Spectrom. 1998, 9, Analytical Chemistry, Vol. 78, No. 7, April 1,

4 approximately a s scan cycle time, wherein ion filling of the linear trap and ion transfer take a significant share of this time. Measured values of resolving power are always higher than the nominal value. Typical AGC target values N are for the linear trap detector and for the orbitrap detector. With the use of an electrometer, it was determined that the number N is actually quite close (within factor of 2) to the real number of ions stored in the linear trap. Mass calibration coefficients are determined for four different AGC target values and interpolated for intermediate values. No magnitude-dependent corrections of m/z are made for data processing. Initial calibration of the instrument is performed using the standard LTQ calibration mixture with caffeine, the peptide MRFA, and Ultramark 1600 dissolved in 50:50 v/v. water/acetonitrile solution. Bradykinin, bovine serum albumin, horse heart apomyoglobin, horse cytochrome c, and carbonic anhydrase proteins were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany) and used without further purification. A Finnigan Surveyor Plus liquid chromatograph (Thermo Electron, San Jose, CA) fitted with a Hypersil C18 column (2.1-mm i.d., 5-µm particles, Thermo Electron) was used for LC/MS runs. Nanospray emitters from Proxeon (Odense, Denmark) were used for static nanospray experiments. PicoFrit columns (75-µm i.d., New Objective, Woburn, MA) were used for nano-lc/ms. During experiments, special care was taken handling caffeine, TFA, and acetonitrile according to the manufacturer s guidelines. Figure 2. Wide-mass range, single-scan mass spectra of calibration mixture in the linear trap (a) and in the orbitrap at nominal resolving powers of (b) 7500 and (c) at AGC target value N ) with external calibration. Inset shows a minor doublet of two isotopomers ( 34 S and 13 C 2) of MRFA peptide separated by a theoretical mass difference Da. with Xcalibur software. Mass spectral data can be stored in fullprofile or reduced-profile format. In the latter, data below the threshold of detection (i.e., close to the thermal noise of the preamplifier) are removed to reduce the size of the dataset. The resolving power of the orbitrap (full width half-height) is switched in discrete steps between the following nominal values at m/z ) 400: (1.9-s scan cycle time), (1-s scan cycle time), , and The latter corresponds to RESULTS AND DISCUSSION Resolving Power. Figure 2 demonstrates wide-mass range, single-scan mass spectra acquired with different resolving powers. Intensity distributions are similar at the two resolving powers (R ) 7500 and R ) ) and absolute abundances differ by <20%. This reflects the fact that resolving power is determined by the acquisition time and not by orbitrap imperfections such as inaccuracy of manufacturing or insufficient vacuum. It should be noted that the resolving power is also unaffected by the AGC target value. Because trapping in the orbitrap is electrostatic (i.e., no magnetic field and no rf potentials), the frequency of the axial oscillations is inversely proportional to the square root of m/z, 7 in contrast to the cyclotron frequency in FTICR, which is inversely proportional to m/z. As a result, for a fixed acquisition time, the resolving power of the orbitrap mass analyzer diminishes as the square root of m/z, i.e., slower than in FTICR (though it should be noted that the resolving power of the orbitrap remains lower than that of 7-T FTICR until m/z 900 and to m/z 2500 of 12-T FTICR). As previously observed, 9 the resolving power of the orbitrap diminishes with an increase of ion mass, even if m/z is unchanged. This effect has been attributed to collisions with background gas that lead to fragmentation of ions and formation of noncoherent packs of fragment ions. For a given m/z, the center-of-mass collision energy with residual gas remains the same while the collision cross section increases with mass, thus leading to faster scattering, fragmentation, and transient decay. This process is more pronounced in the orbitrap than in FTICR because the ion energy is independent of m/z in the orbitrap, while in FTICR it decreases as (m/z) -1. Thus, ultrahigh vacuum is critical for high Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

5 Figure 3. Measured and theoretical isotope distributions of proteins at setting R ) (100 scans averaged, external calibration): (a) horse heart apomyoglobin, m ) Da (monoisotopic, neutral), z ) +10; (b) carbonic anhydrase, m ) Da (monoisotopic, neutral), z )+21. resolution measurements of proteins. Figure 3 presents examples of high-resolution spectra of higher-mass proteins acquired at the same settings as Figure 2b. Although the resolution of these proteins is inferior to that for singly charged ions of the same m/z (Figure 2), it remains sufficient to correctly resolve the isotopic envelope and to provide good mass accuracy for both of the proteins ( ppm). Mass Accuracy. A two-point calibration is used to calculate mass in the orbitrap, even though it is only an approximation for a more complex calibration law. This can be seen in Figure 4, where mass traces for different ions (those in Figure 2) are drawn as a function of AGC target number N with MRFA at m/z ) 524 serving as an internal calibrant. It is clear that despite widely varying peak intensities within and between mass spectra, internal calibration mass accuracy stays well within 2 ppm even for extremely large numbers of ions (i.e., N > 10 6 ). With even larger ion populations, space charge effects in the C-trap distort energy and spatial distributions to such an extent that this starts to affect ion distribution and movement inside the orbitrap. Figure 5 shows the long-term stability with external calibration, which demonstrates that mass accuracy stays well within 5 ppm over 15 h. Variability is due to shot noise and thermal sensitivity of the inner electrode voltage, which is greater than mass shifts due to space charge effects. To optimize external calibration stability, the orbitrap and associated power supplies are thermally regulated. Transmission and Detection Limit. Signals measured with the linear trap and orbitrap detectors were compared in order to estimate the transmission. This comparison was carried out at a very low AGC target value N ) 200 using spectra shown in Figure 6. The full-profile orbitrap spectrum shows the noise band produced by thermal noise of the image current preamplifier. It should be emphasized that linear trap detection employs electron multipliers capable of single-ion detection while image current detection of the orbitrap has an inherent noise band equivalent to 20 ions (for 1-s acquisition length), as estimated using a previously published method. 14 Such a relatively small noise band was achieved by optimizing the preamplifier design as well as minimizing the length of signal wires. As linear trap noise peaks are clearly single ions of chemical background, this could be used to estimate the total number of ions at m/z ) in Figure 6a: ions total in 10 scans (or ions in a single scan). Given that the ion transmission from the axis of the linear trap to its detectors is better than 50%, 6 this brings the original number of ions inside the linear trap to for N ) 200. For the mass peak at mass m/z ) 524, the signal-to-noise ratio on the orbitrap is S/N ) 13 for 10 averaged scans, which results in S/N ) 4 for a single spectrum. The average S/N ) 4inthe orbitrap at noise band equivalent of 20 ions yields an estimation for an average number of detected ions of (taking into account isotopes). This indicates a transfer efficiency from the linear trap to the C-trap and then to the orbitrap of 30-50% (with the first number corresponding to m/z < 200 and the second to m/z > 1000), which is consistent with independent direct electrometer measurements at higher target numbers N (data not shown). To estimate detection limits, direct measurements of a lowconcentration solution of bradykinin were carried out in static (14) Limbach, P. A.; Grosshans, P. B.; Marshall, A. G. Anal. Chem. 1993, 65, Analytical Chemistry, Vol. 78, No. 7, April 1,

6 Figure 6. Comparison of 10 averaged spectra for (a) the LTQ and (b) the orbitrap detectors for the same very low AGC target N ) 200 and a narrow mass range containing MRFA peptide (m/z ) ) (R ) in the orbitrap, full-profile mode, external mass calibration). Figure 4. Mass errors plotted for different m/z as a function of AGC target value N with the mass peak of MRFA peptide (m/z ) ) used as an internal mass calibrant at R ) : (a) m/z ) , (b) m/z ) , and (c) m/z ) Figure 5. Long-term mass stability for MRFA (m/z ) ) using external mass calibration (R ) , N ) , single scan acquired every 6 s over 15 h). Figure 7. Mass spectrum of low-concentration bradykinin (charge state +2, theoretical monoisotopic m/z ) ) on the LTQ orbitrap (3 amol consumed, 3 nm sample concentration, R ) , N ) 5000, 1.16-s injection time and 2-s total scan cycle time, external mass calibration). nanospray mode using a nanospray ion source at 30 ( 10 nl/ min flow rate. Freshly prepared stock solutions of bradykinin at 100 fmol/µl were serially diluted to a concentration 3 fmol/µl. Figure 7 shows a spectrum with 3 amol of bradykinin consumed over the duration of a 2-s scan (R ) ). Dynamic Range. The dynamic range over which accurate mass measurements can be made is a key figure of merit as it actually determines the utility of accurate-mass capability for reallife applications much more than other parameters (e.g., resolving power). While detailed consideration is to be published elsewhere, Figure 8 provides an example of the dynamic range of mass accuracy of the LTQ orbitrap for real-life HPLC-MS analysis and shows that mass accuracy is achieved in a single scan even for mass peaks with up to 5000 times difference in abundances (peak at m/z ) 260 vs peak at m/z ) 610). The spectrum in Figure 8 was acquired in the reduced profile mode Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

7 Figure 8. Illustration of dynamic range of mass accuracy of the LTQ orbitrap (siloxane impurities in propanolol sample) in a single 1-s scan (R ) , N ) , external mass calibration, reduced profile mode). Figure 9. Example of data-dependent acquisition with external mass calibration for a sample containing small molecules, with one highresolution mass spectrum recorded of the precursors at R ) and N ) (a) followed by three data-dependent MS/MS spectra at R ) 7500, N ) , (b) for precursor at m/z ) 260, (c) for precursor at m/z ) 310, and (d) for precursor at m/z ) 386. Data-Dependent Acquisition. Figure 9 is an example of rapid data-dependent acquisition for a sample containing small molecules using a fast gradient (3 min from 100% A to 100% B, where A is water with 0.1% TFA and B is acetonitrile with 0.1% TFA) LC separation on a Hypersil C18 column (2.1-mm i.d., 5-µm particles). A mass spectrum at nominal resolving power of (scan cycle time 1 s) is followed by three rapid data-dependent MS/MS acquisitions at nominal R ) 7500 at a rate of 2.5 spectra/s. Fast acquisition of MS/MS spectra is possible because of the high transmission between the linear trap and the orbitrap, which allows shorter ion accumulation times than in FTICR and TOF. Despite lower resolving power settings in MS/MS spectra, mass accuracy is not sacrificed so elemental composition can be unambiguously determined in all cases. CONCLUSION The main performance parameters of a novel linear ion trap/ orbitrap hybrid mass spectrometer have been characterized. A novel approach to ion storage and injection into the orbitrap allows high resolving power, mass accuracy, and transmission over a wide dynamic range and forms the basis for a hybrid mass spectrometer combining these analytical parameters with the MS n capability of the linear ion trap mass spectrometer. Utilizing short fill times of the linear trap and relatively low number of ions for Analytical Chemistry, Vol. 78, No. 7, April 1,

8 analysis in the orbitrap, this hybrid is capable of providing rapid, accurate-mass MS n analysis of complex mixtures with external calibration. ACKNOWLEDGMENT The authors express their deep gratitude to colleagues who made invaluable contribution to the development of this instrument: Dr. Reinhold Pesch, Dr. Gerhard Jung, Frank Czemper, Oliver Hengelbrock, Silke Strube, Dr. Hans Pfaff, Dr. Torsten Ueckert, Ralf-Achim Purrmann, Juergen Srega of Thermo Electron (Bremen) GmbH, Dr. Mike Senko, Mark Hardman, Mike Antonczak, Dr. Eric Hemenway of Thermo (San Jose), Dr. Steve Davis, Robert Lawther, and Andrew Hoffmann of HD Technologies Ltd. and later Thermo Masslab Ltd. The authors thank Dr. Mike Senko for valuable comments on the manuscript. Received for review October 20, Accepted February 1, AC Analytical Chemistry, Vol. 78, No. 7, April 1, 2006