Anal. Chem. 2002, 74, 1402-1407
A Tandem Mass Spectrometer for Improved Transmission and Analysis of Large Macromolecular Assemblies Frank Sobott, Helena Herna´ndez, Margaret G. McCammon, Mark A. Tito, and Carol V. Robinson*
Cambridge University Chemistry Department, Lensfield Road, Cambridge CB2 1EW, U.K.
We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3000-4000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22 000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90 000, in theory to more than m/z 150 000. We also demonstrate that the resolution of the instrument is greater than 3000 at m/z 44 500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer. The past decade has seen the widespread application of electrospray ionization (ESI) mass spectrometry not only as a tool for the analysis of individual molecules but also, more recently, macromolecular assemblies of increasing size and complexity.1-3 ESI is unique in that it provides for a relatively gentle transition of analyte from the sample solution into highly charged droplets and, finally, to molecular ions in vacuo by a combination of thermal and collision-induced desolvation.4-6 When electrospray is carried * Phone: 44 1223 763844. E-mail:
[email protected]. (1) Rostom, A. A.; Robinson, C. V. Curr. Opin. Struct. Biol. 1999, 9, 135-141. (2) Loo, J. A. Int. J. Mass Spectrom. 2000, 200, 175-186. (3) Herna´ndez, H.; Robinson, C. V. J. Biol. Chem. 2001, 276, 46685-46688. (4) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 240, 64-70. (5) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. (6) Du ¨ lcks, Th.; Juraschek, R. J. Aerosol Sci. 1999, 30, 927-943.
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out from acidified solutions containing organic solvent, a series of highly charged ions is produced for macromolecules. This has the inherent advantage that the multiply charged ions can be separated according to their m/z values in analyzers with limited m/z range, for example, quadrupoles. Such analyzers routinely operate up to m/z 3000 to 4000 and can tolerate the relatively high pressures associated with ESI. With the introduction of nanoflow electrospray,7 the smaller size of the initial droplets facilitates the use of pure aqueous solutions and more gentle desolvation conditions such that noncovalently bound complexes are preserved more readily than when conventional ESI is employed.8 Functional assemblies of biomolecules introduced from solutions in which their native state is preserved often give rise to signals well above m/z 4000.9 For large macromolecular particles, m/z values of over 20 000 have been recorded.10,11 Although some examples have been reported that demonstrate tandem-MS of complexes containing multiple copies of a single protein,9,12 much more information can be obtained for large heterogeneous assemblies by this approach. Conventional tandem mass spectrometers have limitations for studying such complexes, since the first mass analyzer is usually a quadrupole with limited m/z range. In addition, the different pressure regions of the mass spectrometer are not optimized for the transmission of large macromolecular complexes, which require gentle desolvation conditions. As the gas and the still partly solvated ions expand adiabatically through the orifice from atmospheric pressure into vacuum, they are internally cooled and a molecular beam is formed.13,14 The speed and off-axis movement of the ions are dampened by collisional cooling.15 To survive the transition from the solvated (7) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (8) Chung, E. W.; Henriques, D. A.; Renzoni, D.; Morton, C. J.; Mulhern, T. D.; Pitkeathly, M. C.; Ladbury, J. E.; Robinson, C. V. Protein Sci. 1999, 8, 1962-1970. (9) Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. J. Am. Chem. Soc. 1994, 116, 5271-5278. (10) Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5185-5190. (11) Tito, M. A.; Tars, K.; Valegard, K.; Hajdu, J.; Robinson, C. V. J. Am. Chem. Soc. 2000, 122, 3550-3551. (12) Tahallah, N.; Pinkse, M.; Maier, C. S.; Heck, A. J. R. Rapid Commun. Mass Spec. 2001, 15, 596-601. (13) Fenn, J. B. Int. J. Mass Spectrom. 2000, 200, 459-478. (14) Thomson, B. A. J. Am. Soc. Mass Spec. 1997, 8, 1053-1058. (15) Krutchinsky, A. N.; Chernushevich, I. V.; Spicer, V. L.; Ens, W.; Standing, K. G. J. Am. Soc. Mass Spectrom. 1998, 9, 569-579. 10.1021/ac0110552 CCC: $22.00
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state in the bulk liquid to a “naked” existence in the gas phase, the energy input required for the phase transfer should not exceed the dissociation threshold of the complexes. The energy input can be controlled by careful adjustment of the acceleration voltages in the different stages of the instrument. The analyte undergoes collisions with background gas atoms, by which it is activated and subsequently loses residual, attached solvent molecules. The amount of activation per collision depends on the kinetic energy of the ions, which is determined by the accelerating electric field as well as the average collision frequency with background gas atoms according to the pressures in the different stages of the instrument. We - and others - have observed that raising the background pressure in the initial vacuum stages favors the transmission of high-m/z species and appears to be a prerequisite for the detection of intact noncovalent complexes12,15,16. By fitting an additional leak valve at the second pumping stage, we observed that additional collisional cooling in the RF hexapole lens assembly provides considerable increases for the ion intensity of macromolecular complexes. In tandem mass spectrometry, also known as MS/MS or MS2, a wealth of information about molecular structure and stability of covalent molecules has been obtained.17,18 Ions selected according to their m/z ratio in the first mass analyzer undergo collisioninduced dissociation (CID) in a gas-filled collision cell.19 The subsequent MS stage allows analysis of the dissociation products that are formed. In the Q-TOF, the first stage is a quadrupole and the second, a time-of-flight (TOF) analyzer. Although the mass range of a TOF is in theory unlimited, quadrupoles can transmit ions only to a certain upper limit, which is dependent on the RF amplitude and frequency as well as the inscribed diameter of the rod assembly. The highest m/z, Mmax, that can be transmitted is given by
Mmax )
7 × 106 Vm f 2r20
(1)
with Vm cos(2πft) as the RF voltage applied between adjacent rods (2Vm is the peak-to-peak amplitude; f, the frequency; and t, the time) and r0 as the inner radius between rods in meters.20 For a standard Micromass Q-TOF, which operates at a frequency of 832 kHz, the mass range of that first stage is limited to m/z 4190. To increase the upper transmission limit, in principle, all three parameters (Vm, f, and r0) in eq 1 could be changed. Vm is limited because of possible breakdown of the high voltage between the rods, and decreasing r0 can reduce transmission and also the tolerance to mechanical misalignment and surface contamination. There is, however, no practical lower limit to the frequency, so that by adjustment of this parameter, the mass range can be extended indefinitely. Using this approach, single quadrupole mass spectrometers have been designed with a m/z range of up to (16) Rostom, A. A.; Robinson, C. V. J. Am. Chem. Soc. 1999, 121, 4718-4719. (17) de Hoffmann, E. J. Mass Spectrom. 1996, 31, 129-137. (18) Shukla, A. K.; Futrell, J. H. J. Mass Spectrom. 2000, 35, 1069-1090. (19) Jennings, K. R. Int. J. Mass Spectrom. 2000, 200, 479-493. (20) Austin, W. E.; Holme, A. E.; Leck, J. H. In Quadrupole Mass Spectrometry and its Applications, 1st ed.; Dawson, P. H., Ed.; American Institute of Physics: Woodbury/New York, 1995; Chapter 6.
900021 and 45 000.22 The resolution that could be obtained with these instruments was, however, significantly reduced when compared to that of a quadrupole operating at higher RF. The highest possible mass resolution is determined by the number of cycles of RF field to which the ions are exposed while they pass through a quadrupole of finite length. The Q-TOF described here uses a custom-built RF unit that operates at a frequency of 300 kHz, while the maximum RF amplitude and rod geometry remain unchanged. Because the quadrupole is used only as a mass filter to isolate ions for tandem-MS and the product ion spectrum is acquired using a TOF analyzer, the resolution is not compromised significantly by the change in operating frequency of the quadrupole. During the design of this instrument, we explored two critical factors for transmission and mass analysis of macromolecular complexes: the first, that of the operating pressure in the different regions of the mass spectrometer, governed by the orifice dimensions, vacuum pumps, and provision of gas inlets for collisional cooling; and the second, the m/z range of the first mass analyzer and its effect on the overall mass resolution of the instrument. We demonstrate the capabilities of the modified Q-TOF using cesium iodide cluster signals, where peaks beyond m/z 90 000 could be detected with only moderate reduction in mass resolution. We show that individual species can be isolated in the quadrupole at m/z 22 000 (in theory up to m/z 32 000). Using the complexes formed between transthyretin, thyroxine, and retinol binding protein,23 we demonstrate the transmission of noncovalent biomolecular complexes and the MS/MS of an individual charge state from the resulting heterogeneous mass spectrum. EXPERIMENTAL SECTION Apparatus. The basis for the extended m/z range tandem mass spectrometer is a Q-TOF 2 instrument (Micromass, Manchester, U.K.)24 with a Z-spray source. The Q-TOF 2 has been modified in several respects (Figure 1), as discussed below. Sample solutions were introduced either by conventional electrospray against a counter-flow of dry, pure nitrogen gas (flow rate 100200 L/h) or by nanoflow electrospray using gold-coated borosilicate glass capillaries prepared in-house. Partially solvated ions are transferred through a sample cone with an orifice of 400 µm oriented at 90° to the direction of the electrospray into the first vacuum stage of the instrument (p1 in Figure 1). The molecular beam that evolves behind the sample cone is then extracted orthogonally through a skimmer (extractor cone) into the second pumping stage (p2). The pressure p1 between the two cones is regulated with a speedivalve (BOC Edwards, Crawley/Sussex, U.K.), that throttles pumping by the rotary pump, and the acceleration voltage is varied in the range 50-200 V for optimum transmission and resolution in the m/z range of interest. Ions entering the second pumping stage (p2, Figure 1) are focused by (21) Labastie, P.; Doy, M. Int. J. Mass Spectrom. Ion Processes 1989, 91, 105112. (22) Winger, B. E.; Light-Wahl, K. J.; Ogorzalek-Loo, R. R.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 536-545. (23) Rostom, A. A.; Sunde, M.; Richardson, S. J.; Schreiber, G.; Jarvis, S.; Bateman, R.; Dobson, C. M.; Robinson, C. V. Proteins, 1998, Suppl. 2, 3-11. (24) Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Commun. Mass Spec. 1996, 10, 889-896.
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Figure 1. Schematic layout of the modified Micromass Q-TOF 2 instrument. The ion beam is indicated by a black line of varying width. For a description of the instrument and the modifications, see the Experimental Section.
a hexapole ion guide (RF lens). A DC voltage offset between the cone and the hexapole rods of 0-50 V determines the ion acceleration when they enter this stage. Here, collisional dampening of the ion movement takes place at pressures p2 in the range of 3 × 10-4 to 1 × 10-2 mbar. The modified instrument has been fitted with an additional leak valve for admitting argon so that the pressure in this region can be increased and optimized. Although the precise relationship between ion transmission and pressure in various regions of the Q-TOF is not known in detail, our experimental approach is to start the acquisition at base pressures and then adjust the pressure in each stage of the instrument sequentially, starting with the source region, for optimal signal. Thus, we found that operating this instrument in all stages at pressures that are higher than in a normal Q-TOF considerably improves ion transmission at high m/z. Ions exit the RF lens with a kinetic energy determined by the voltage offset to the next aperture (“ion energy”, usually
