Anal. Chem. 1998, 70, 647R-716R
Review
Mass Spectrometry A. L. Burlingame*
Department of Pharmaceutical Chemistry, The Mass Spectrometry Facility and the Liver Center, University of California, San Francisco, California 94143-0446 Robert K. Boyd
Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada B3H 3Z1 Simon J. Gaskell
Department of Chemistry, University of Manchester Institute of Science & Technology, Manchester, U.K. Review Contents Overview Scope Innovative Techniques and Instrumentation Isotope Ratio Mass Spectrometry High-Power Lasers in Mass Spectrometry Dissociation by Low-Intensity Infrared Radiation Polymers Peptides and Proteins Oligonucleotides and Nucleic Acids Literature Cited
647R 648R 652R 668R 673R 677R 679R 681R 692R 698R
OVERVIEW Viewed from the centenary of the discovery of the electron by J. J. Thomson (A1), it might be expected that the principles and current technologies employed in the detection and manipulation of charged species would be sufficiently mature to tackle even the most challenging scientific problems. This could not be further from the truth in studies of human biology, as we contemplate the demands involved in dissecting the myriad macromolecular details involved in understanding homeostasissthe expression, regulation, and concerted function of some 10 000 proteins per cell. Such a comprehensive understanding must eventually be achievable at the level of a single normal or somatic cell. Over this past decade mass spectrometry has finally escaped the earlier constraint that required volatilization of substances and has become a suite of powerful, versatile, and sensitive tools essential for solving the problems of low-level protein identification and structural characterization. These technologies have reached the threshold of addressing the delineation of composition and architecture of protein and other macromolecular machines (A2), thus setting the stage for the Millikan “water drop” experiment with mammalian organelles and cells in mind. The fundamental processes at play in effecting the most powerful desorption/ionization phenomena known to date, MALDI and electrospray, will eventually yield to mechanistic understanding. Surely, additional ionization and detection methods will be discovered. More powerful sample manipulation and ion optical S0003-2700(98)00023-7 CCC: $15.00 Published on Web 07/09/1998
© 1998 American Chemical Society
strategies will emerge to address the sheer size and complexity of molecular machines, as well as enable scrutiny of the makeup of single human cells and their environmental signals. For the present, the delayed extraction MALDI and nanoflow electrospray technologies together with a new generation of highperformance instrumentation are just beginning to add much needed analytical power to our arsenal for macromolecular identification and structural characterization, at reasonable cost. These techniques are based on optimized time-of-flight (TOF) technologies, but solenoid-based FTMS systems are gaining ground as well. Already these instruments are at work in laboratories that have had no previous experience with the clarity, precision, and structural definition that mass spectrometric technologies can bring to biological research. This trend will bring an awareness of the importance of these methods to biological and clinical scientists and thus accelerate the integration of mass spectrometry into biomedical research strategies. Much of the current literature is concerned with characterization of proteins and their posttranslational or xenobiotic modifications, as well it should be, as proteins make up most of the dry mass of a cell (A2). The focus is on the discovery of unknown protein function and the delineation of the biosynthesis and processing of physiologically functional forms of proteins, their regulation, degradation, and use in immune surveillance. The focus is also on understanding the immmediate environment of a cell and the modulation of its homeostasis by endogenous or environmental agents. In addition, our knowledge of protein identity and function is lagging far behind our knowledge of several genomes, leading to a recent call to redouble our investment in biomedical research so that we may realize the fruits of molecular medicine by 2020 (A3). This call recognizes overtly that new technologies, tools, and instrumentation must be invented to support and enable this research challenge (A4) to realize individualized medicine in the next quarter century. The need to focus attention on gaining this essential knowledge of the machinery of cells has been articulated recently as the next national goalsthe biomedical “moon shot”: “...The 21st Century Research Fund will give us the means to win Analytical Chemistry, Vol. 70, No. 16, August 15, 1998 647R
the war on cancer. It will allow the development of new classes of smart drugs that target specific molecules found in cancer cells. It will help researchers discover, within a decade, every single gene and protein that contributes to the conversion of a normal cell to a cancer cell...” (A5). Mass spectrometry is destined to play a pivotal role in this endeavor. SCOPE The sections of these reviews labeled “Scope” have traditionally been used for several purposes, including delineation of which subdisciplines and applications are to be covered and (equally important) which have had to be excluded for reasons of time and space. In this regard we offer our usual apologies for all our sins of omission and commission. The burgeoning developments and applications in mass spectrometry are a tribute to the creativity and energy of our colleagues, but make it increasingly difficult to provide comprehensive coverage in these biennial reviews. Accordingly, we have tried to provide continuing coverage of the most active areas, particularly biological applications (especially to peptides and proteins including proteomics, and to nucleic acids) and development of instrumentation and methodology designed for such purposes [including mass analyzers, detectors, the new ionization techniques of electrospray and matrix-assisted laser desorption and ionization (MALDI), and direct coupling of mass spectrometers to separation techniques, with some emphasis on issues associated with quantitation and automation to provide high-throughput analyses]. In addition, less frequent vignettes have dealt with other important but (as yet) less widespread activities. In the present review these special topics include isotope ratio mass spectrometry, combination of mass spectrometry with fast (nanosecond) and ultrafast (femtosecond) lasers, interactions of ions with low-intensity infrared radiation including ambient blackbody radiation, and applications to synthetic polymers. Aspects notably absent from the present review include explicit treatments of more fundamental aspects, mass spectrometry of small molecules (30 000. Hiroki et al. (C7) investigated the effect of a prefilter on the transmission at m/z 4 of a quadrupole operating near (3.1, 3.2). These investigations (C6, C7) have applications in plasma physics and include references to earlier work in this area. More recently, Du et al. (C8) designed a quadrupole for use with an inductively coupled plasma source for elemental analysis up to m/z 78 and which was operated near the (3.1, 3.2) stability region. A limiting resolution (based on peak fwhm) of 4000 was obtained at m/z 59, insufficient to separate atomic ions from interfering molecular ions of the same nominal mass except in favorable cases. However, the abundance sensitivity at moderate resolution (500-1000) was very high, and this approach was suggested to be useful for specialized applications. Ying and Douglas (C9) later described a system designed for the same purpose, but operating near (0.03, 7.55), which successfully resolved Fe+ from ArO+ while maintaining sensitivity comparable to that of double-focusing analyzers for ICP operated at the same resolution, although the background was much higher. It was possible to switch quickly between stability regions, thus trading resolution for sensitivity. Other characteristics of this same stability region (requirement for fewer rf cycles to achieve mass resolution, thus permitting analysis of fast ion beams) were exploited for a different purpose by Grimm et al. (C10), who operated at a ) 0 (rf-only mode) in order to
achieve fast scanning (80 000 m/z units/s) for high-speed gas chromatography. This amounts to 1000 scans/s for the current range up to m/z 80, but with several disadvantages (C10) including decreased sensitivity and so-called “mass-aliasing”, in which ions with m/z values 8.25 times that of the ions of interest are stable in the first stability region and are transmitted as artifacts. Hiroki et al. (C11) used the same (0, 7.55) stability region to analyze fast ion beams with energies up to 3 keV. For quadrupoles operated in the first stability region, an unusual operating scan mode using rf only, i.e., at the (0, 0.906) edge of the first stability region, relies on the strong focusing fields at this operating point as first suggested by Brinkmann (C12). It is well-known that an rf-only quadrupole acts as a high-pass mass filter, but Brinkmann realized that ions whose trajectories correspond to q values near the critical cutoff value (0.908) will exit the quadrupole preferentially near the edge of the aperture circle and will experience a net acceleration in the fringe fields. Such higher energy ions will pass through a lens or screen at a potential that will reject the beam of stable ions (q < 0.908) still constrained to move close to the axis, and thus experiencing little or no net acceleration upon exiting. Therefore by applying a dc retarding field between the quadrupole exit and the detector, the high-pass filter mode is transformed into a mass resolving mode with useful resolution and transmission. This concept was reviewed by Weaver and Mathers in 1978 (C13) and was recently revived by Pedder and Schaeffer (C14, C15) who demonstrated its utility for analysis of ions at higher m/z values, including cluster ions and large biomolecules. This same objective was achieved in a different manner by Collings and Douglas (C16), who increased the mass range of a conventional quadrupole mass filter (operating near the tip of the first stability region) by dropping the rf frequency. This is a well-known approach, but by restricting the frequency decrease to ∼70%, only minor changes in sensitivity were observed while doubling the m/z range and providing unit mass resolution up to m/z 5000 (C16). A new scanning mode for operating near the tip of the first stability region, by scanning the rf frequency while maintaining both the rf and dc amplitudes constant, has been described by Landais et al. (C17). Only preliminary results are available thus far, since the resolution is currently limited by the amplitude available from the variable-frequency rf supply. Theoretically the resolution (rather than the peak width) should be independent of m/z using frequency scanning (C17). Miniature quadrupole arrays, which can operate at higher operating pressures (10 mTorr) have been constructed for residual gas analysis (C18), and the performance of such devices has been demonstrated (C19). A method for mounting even smaller quadrupole arrays on a single chip by silicon etching (C20) has been described, but thus far no performance data appear to have been published. A novel rf band-pass mass filter has been designed (C21) for highcurrent ion beams, which are broad, to keep space charge effects under control. A careful evaluation of the m/z dependence of the transmission efficiency of a quadrupole mass spectrometer has been described (C22), but unfortunately, the contributions of the mass filter alone to the discrimination could not be unambiguously extracted. An ingenious method of selective collisional activation of ions in a gas-filled (1 mbar) rf-only quadrupole with a controllable axial field, by selective acceleration by the rf field
near the stability limit (q ) 0.906), has been described by Dodonov et al. (C23). Several investigations of quadrupole performance by computer simulation have been published. Yoshinari et al. (C24) used a boundary element method to calculate the field and a high-order Runge-Kutta method to calculate ion trajectories with modest demands on computer memory and time. Takebe and Kumashiro (C25) used a trajectory simulation program to investigate fringefield effects. Reuben et al. (C26) developed exact coefficients for the multipole potential expansion in a quadrupole constructed from cylindrical rods in a grounded casing and, as a result, suggested a repositioning of the mass scan line on the first stability region. Tunstall et al. (C27, C28) have developed a comprehensive computer simulation package for quadrupole mass filters and have investigated effects of aperture, rf harmonics, and finite rod length. Ioanoviciu (C29) developed analytic expressions for ion trajectories inside a quadrupole in terms of trigonometric series with timedependent amplitudes. These solutions are valid only in the neighborhood of the tip of the first stability region, but suggest that some particular ion trajectories predicted to be unstable by more conventional treatments should in fact result in transmission through the mass filter (C29). Titov (C30, C31) has developed a comprehensive analytical approach, based on a phase-space treatment of beam dynamics, of quadrupoles operating in all three stability regions. Using this approach it was possible to elucidate the phase dependence of the ion transmission which is therefore modulated at the rf frequency (C30). These losses can be reduced by applying a standing wave potential with a linear amplitude distribution along the length of the quadrupole, and it is proposed (C31) that this principle could be exploited to significantly reduce the required mechanical tolerances and to increase the attainable resolution in quadrupoles. A high-transmission pentaquadrupole instrument designed for mutidimensional tandem mass spectrometry has been described by Eberlin et al. (C32, C33). (b) Paul Traps. The Paul trap (rf quadrupole trap) was originally developed by physicists interested in increasing the observation time available for spectroscopic measurements on elementary particles, thus reducing the inherent uncertainty in energy measurements via Heisenberg’s uncertainty principle, as outlined by Holzscheiter (C34) in a brief history of such devices from the physicists’ point of view. The experiments described by Holzscheiter (C34) are of mainly historical interest to analytical chemists, but it is interesting to read the quotations from early workers stating that it is impossible to trap ions injected from an external source. An excellent introduction to ion traps in analytical chemistry, directed principally at biochemists, has been published recently by Jonscher and Yates (C35). Before proceeding to discuss recent innovations in rf quadrupole traps, however, attention is drawn to a remarkable innovation due to Benner et al. (C36, C37). This innovation consists of an electrostatic trap involving no rf or magnetic fields, which permits simultaneous measurement of ion charge (via image charge current) and time of flight of ions of known translational energies. The original implementation (C36) used a single pass of the ions, but more recent improvements (C37) permit remeasurement on the same ions more than 450 times, to greatly improve the signalto-noise ratio and the consequent precision of measurement of both mass and charge of very large ions, e.g., 2.88-MDa ions of Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
653R
DNA carrying more than 250 charges. This novel technique was developed to determine the mass of plasmid DNA, but other possible applications include identification of overlap in clones, chromosome sorting, very large noncovalently bound subcellular structures, and virus particles and bacterial spores (C37). Since the remarkable advances in Paul trap methodology reported in the 1996 edition of this review, further progress has been made on a number of fronts. As normally employed, rf ion traps record a mass spectrum using the mass-selective instability method devised by Stafford et al. (C38), in which ions of different m/z values are progressively ejected and detected using a conventional electron multiplier. Such a destructive detection mode (common to most other mass spectrometers) provides in principle considerably higher detection limits than do strategies using nondestructive detection, which permit measurement of a given ion population many times. This nondestructive strategy is commonplace in FTICR practice but until recently was almost unknown in chemical applications of Paul traps, although early workers including Paul himself had employed nondestructive detection techniques for narrow m/z ranges. Recently narrowband nondestructive detection in Paul traps was demonstrated by Parks et al. (C39) and by Goeringer et al. (C40). Extension to broad-band frequency (and thus m/z) detection, using Fourier transform analysis of image currents produced by coherently excited ion populations, has been described by Soni et al. (C41). An important innovation in this work (C41) was the incorporation of a small detector electrode, imbedded in the end cap but isolated electrically from it, used as an antenna for the image currents. Some critical background work related to this important advance included multiparticle simulation studies of short-burst ion injection using dc dipolar fields to supplement the damping provided by helium buffer gas (C42) and development of a program permitting visual representations of simulated ion trajectories in a Paul trap (C43). The effects of deviations of the trap potential from the ideal form assumed in first-order theory are well established, e.g., the “black hole” first reported by Guidugli and Traldi (C44, C45) and further characterized by Eades et al. (C46). In a series of papers (C47-C49) Alheit et al. have investigated the effects of nonlinear resonances caused by nonideal field contributions, arising from causes such as deviations from hyperbolic shape of the electrodes, truncation of the electrodes (hyperbolas extend to infinity), or space charge. Their observations were largely in accord with theoretical treatments of Wang et al. (C50-C52), although some additional effects thus far unexplained were also observed. Doroshenko and Cotter (C53) investigated the effects of these higher-order fields on ion losses during forward and reverse scans and suggested methods for alleviation of these ion loss effects. Field imperfections can also give rise to significant mass shifts which are effectively eliminated by stretching the spacing of the end caps (C54) or by coupling of motions of ions of closely similar m/z values, so-called “local space charge” effects (C55). Many of the latter observations (C55) have been further investigated and interpreted by Mo and Todd (C56), who suggest that the ion-ion coupling effect is one of the most important factors that influence the accuracy of mass assignment in high-resolution operation of ion traps. A different kind of ion-ion coupling in Paul traps concerns simultaneously trapped positive and negative 654R
Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
ions. Luo et al. (C57) discussed the correlated motion of two oppositely charged cooled ion clouds. Stephenson and McLuckey (C58) investigated the effects on high m/z cations of a cloud of anions of much higher total charge in the trap center. For example, high m/z cations can be trapped in the field of such a cloud when the trapping quadrupole field is unable to do so. However, the anion cloud interferes with the resonance ejection method for mass analysis, and it is therefore desirable to eject the anions first. These recent findings (C58) confirm and extend some of the earlier experimental findings of Williams and Cooks (C59). Mathurin et al. (C60) investigated the opposite case of the effect of a large excess of positive ions on the low abundance of negative ions formed simultaneously in an ion trap by electron ionization. Again, an enhancement of the storage of the ions of lower abundance (anions in this case (C60)), by the more abundant ions of opposite charge, was observed. A different problem, that of mass assignment and scan-to-scan reproducibility for peak position in a mass spectrum obtained by high-resolution operation (very slow scan rates) of ion traps, was addressed by Londry and March (C61) in an experimental investigation of the effects of phase-locking the fundamental rf and excitation ac frequencies in the resonance ejection operating mode. This approach was further investigated by Doroshenko and Cotter (C62), who showed that the positive effect was appreciable only for higher values of the β parameter. The observations were explained (C62) by negative contributions to the resolution by ion micro-oscillations at the rf frequency along the trajectory. An entirely different approach to investigating motions of ions in an rf trap has been described by Wilhelm et al. (C63), who exploited the fast extraction of ions from a trap operated with no buffer gas into the drift region of a relectron-TOF analyzer (∼500 ns at m/z 100, effectively instantaneous relative to the periodicity of the ion motions in the trap) and subsequent measurement of the ion kinetic energies. The ions were internally cold ions formed by multiphoton ionization of jet-cooled molecules. Variation of storage times caused dramatic variations in ion intensities recorded by the TOF and in the ion kinetic energies (C63). The ions were shown to move in clouds describing Lissajous figures similar to those of a single ion, characteristic of the m/z values, and in the absence of collisions the ion motions remain coherent in space and kinetic energy for up to several milliseconds. Subsequently, Wilhelm et al. (C64) compared such experimental results with trajectory calculations and demonstrated that good mass resolution and stable signal intensity can be achieved in the TOF without the use of buffer gas, provided that ion formation in and extraction from the trap are properly synchronized with the rf field. The combination of jet-cooled precursor molecules with laser ionization and absence of buffer gas permits study of truly unimolecular fragmentation kinetics of ions with well-defined internal energies (C63, C64). Williams et al. (C65) investigated the effects of pulsed introduction of helium buffer gas at various stages of the ion trap operating cycle, instead of the usual constant gas pressure. Introduction of helium just before the ion ejection and detection event was shown to be the most critical and to have an effect independent of the pulse valve open time (i.e., pressure) over a wide range relative to the effect of gas introduced before ionization (C65). Lammert and Wells (C66) investigated the performance of an ion trap using air instead of helium as buffer
gas, an important consideration for field-portable instruments. It was found that resolution and sensitivity depend on the gas pressure and on the q value used for resonance ejection. Air was found to be a satisfactory buffer gas for m/z
