Mass Spectrometry: Recent Advances and Future Directions - The

J. Phys. Chem. 1996, 100, 12897-12910

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Mass Spectrometry: Recent Advances and Future Directions Michael T. Bowers,*,† Alan G. Marshall,*,‡ and Fred W. McLafferty*,§ Department of Chemistry, UniVersity of California at Santa Barbara, Santa Barbara, California 93106; Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Lab, Florida State UniVersity, 1800 East Paul Dirac DriVe, Tallahassee, Florida 32310; and Department of Chemistry, Cornell UniVersity, Baker Lab, Ithaca, New York 14853-1301 ReceiVed: January 12, 1996; In Final Form: March 28, 1996X

The discipline of mass spectrometry is nearly a century old. Like any experimental scientific endeavor, the machines and the science get irretrievably mixed with the development of one stimulating the other in a symbiotic manner. In this paper the authors select a few of the important recent successes to highlight the incredible growth of mass spectrometry over the past several decades. The topic choices made are personal but hopefully serve as an indicator of the current excitement in this rapidly evolving field. Selections are made from nuclear physics, through the broad spectrum of the chemical sciences, to the borders of biology.

Introduction Mass spectrometry, as a discipline, has a rich and fabled history, and it would have been interesting and fun to have chronicled its major achievements as a contribution to this centennial issue of The Journal of Physical Chemistry. Unfortunately, the sheer breadth and depth of the topic made a review of this type far exceed the space allotted for articles in this issue. Consequently, it was decided to greatly reduce the scope of the project and comment only on selected topics developed in the past several decades. In this time frame mass spectrometry has evolved and grown to become one of the most versatile and informative tools for investigation of molecular structure, bonding, and reactivity, particularly for species not (or not yet) readily accessible in condensed phase. For example, the most-cited new molecule of the past decade, buckminsterfullerene (“buckyball”), C60, was first observed and its structure first correctly inferred from mass spectrometric detection and relative abundance of the molecular radical cation, C60•+.1 The current state of the art in mass spectrometry represents a combination of striking recent advances that make it possible to determine ion structure, reactivity, equilibria, and energetics in the gas phase, (i.e., in the absence of solvent). By comparing gas-phase and condensed-phase properties, one can then begin to quantitate and understand the effects of solvent on ion structure and chemistry. The molecular weight range of mass spectrometry has been expanded by several orders of magnitude by new methods for making high-mass ions, particularly laser desorbed/ionized singly-charged polymers2 and supersonic clusters,3 as well as electrosprayed multiply-ionized biological macromolecular ions.4 For example, gas-phase DNA ions of up to 100 000 000 Da have been weighed mass spectrometrically.5 Improved ion optics for focusing and transmitting ions between source and analyzer now make it possible to transport ions almost without loss over distances of a meter or more. Improvement by several orders of magnitude in mass analyzer range, resolution, and mass accuracy makes feasible mass separation and identification of complex mixtures of hundreds of compounds without prior chromatographic separation.6 A host of experiments previously * To whom correspondence should be addressed. † University of California at Santa Barbara. ‡ Florida State University. § Cornell University. X Abstract published in AdVance ACS Abstracts, June 15, 1996.

S0022-3654(96)00154-2 CCC: $12.00

confined to the condensed phase have now become feasible in the gas phase: ion-molecule reactions, drug:receptor binding, hydrogen/deuterium exchange as a probe of protein conformation, biopolymer sequencing, etc. Ions may be detected from as little as an attomole of neutral sample.7 The measurement of atomic masses has reached a precision of better than 1 × 10-9 u, corresponding to a rest mass equivalent energy of ∼1 eV.8 Both the generation and interpretation of mass spectra have been propelled by a variety of theoretical advances: conceptual, analytical, and numerical. For example, gas-phase ion reaction dynamics alone fills an entire separate article in this issue.9 Mass spectrometry, like The Journal of Physical Chemistry, is nearly a century old. In that time incredible advances have been made both in the methods and in its range of applications. It is over the past several decades, however, that near dizzying development has taken place. In this period an unusually fruitful interplay of leapfrog advances in ionization techniques, separation and transport methods, computer technology, and mass analysis methods, driven by applications ranging from crude oil to cosmology, have all propelled the discipline forward. In this brief account, we shall discuss just a few highlights that illuminate part of the mass spectrometry landscape. These are the authors’ personal choices drawn from our interests and experiences. There is much more to mass spectrometry that what is presented here, and the interested reader should seek out the dozens of more technical reviews in Analytical Chemistry A-pages, Accounts of Chemical Research, Mass Spectrometry ReViews, and elsewhere, some of which are cited below. Ultraprecise Mass Measurements Although the direct importance of most problems requiring ultraprecise mass measurement (say, a relative imprecision, ∆m/m ≈ 10-10) derives from fundamental particle physics, many of the methods developed for those problems have been (and continue to be) applied to problems of chemical interest. For example, an ion cooling method adapted from such single-ion physics experiments now makes possible remeasurement of peptide and protein ions (see below). The most precise mass measurements have been based on measurement of the cyclotron frequency of a single trapped ion (or electron) in a Penning trap (see next section for a discussion of ion traps). Typically, one measures the mass difference between two ions detected individually and alternately. Such experiments pose heroic © 1996 American Chemical Society

12898 J. Phys. Chem., Vol. 100, No. 31, 1996 difficulty: (a) fluctuation in atmospheric pressure changes the boiling point of liquid helium and hence the magnetic field in a cryocooled solenoid, so that the boiloff from the helium Dewar must be pressure-regulated to reduce field drift; (b) measurements are typically conducted in the few hours before dawn, to minimize the magnetic field perturbation from subway trains, elevators, etc.; (c) although the cyclotron kinetic energy of a detected ion is typically less than 1 eV, its relativistic increase in mass is significant and must be taken into account; (d) the electrostatic potential field shape must be carefully shimmed to near perfection to avoid systematic errors; (e) the ions must be cooled to liquid-helium temperature before the measurement; (f) ultrahigh sensitivity requires a high-Q detection circuit with a bandwidth of only a few hertz; and (g) ultraprecise radiofrequency stability is required. Here are some examples of the kinds of fundamental phenomena which may be addressed by ultraprecise mass measurements. “Weighing” a Photon. In chemistry, precise mass measurements (to ∼(0.0001 atomic mass units (u)) are routinely used to determine the chemical formula (e.g., CxHyOz‚‚‚) of an unknown molecule. This is accomplished for monoisotopic species by measuring the mass of its corresponding positive or negative ion and by taking into account the ∼0.0005 u mass of the missing or extra electron. From Einstein’s principle, ∆E ) ∆mc2, the rest mass difference, ∆m, corresponding to an energy difference, ∆E, between two quantum states, amounts to about 10-9 u for an equivalent energy difference of 1 eV. Thus, now that it is becoming possible to determine the masses of atomic ions and/or simple molecular ions to an accuracy of better than 1 ppb, one can imagine observing a “chemical” transformation (of say, several electronvolts in energy) by measuring the corresponding change in mass of the “reactant”. In fact, the the mass difference corresponding to the (γ-ray) transition between two nuclear energy states has been measured, a result equivalent to “weighing” a γ-ray photon.10 Similar experiments are currently under way to attempt to “weigh” an optical photon, by measuring the difference between ground and excited states of a rare-gas atomic ion. However, the main value of ultraprecise mass measurements is for testing fundamental issues in modern physics. The most precise mass measurements are presently based on determination of the cyclotron frequency of a single charged particle in a Penning trap. The interested reader is referred to a recent volume of review articles on the subject.11 Electron Mass: Is the Electron a Point Particle? Although we do not usually think of it in such terms, a simple way to tell whether a magnetic particle is “composite” (i.e., is composed of simpler particles and therefore of finite spatial extent) is to measure its “g-factor”, namely, twice the ratio of its Larmor frequency to its cyclotron frequency. For example, Dirac showed in 1928 that if the electron is a point mass, then its g-factor should be exactly 2. (In contrast, the proton g-factor is ∼5.6, which immediately reveals that the proton is composed of subparticles (now known as quarks).) However, a free electron even at “rest” moves in an irregular circle at the speed of light, leading to radiation effects which lead to a small calculable change in the electron g-factor. By confining a single electron in a Penning trap, Dehmelt was able to measure the electron cyclotron frequency as well as its Larmor frequencysthe latter by virtue of spin flips which slightly shift the axial oscillation frequency. After taking quantum electrodynamics corrections into account, the present best experimental value for the electron g-factor still deviates by about 1 part in 20 billion from that computed for a point mass.12 If that difference is real, then the electron is not a “point” particle, but is composed

Bowers et al. of subparticles (denoted by Dehmelt as “subquarks”) whose mass may be computed as ∼10 billion electron masses. If nature were to continue in this way, with sub-subquarks, etc., we would ultimately be left with a single ultramassive “cosmon” particle, which comprised all of the energy and mass of the universe at the “Big Bang”! Resolution of this issue will require even more precise measurements of the electron g-factor (and associated developments in quantum electrodynamics theory). Moreover, many of today’s most accurate frequency standards are based on the ground-state hyperfine structure of hydrogenlike atoms. For example, the S.I. second is defined as 9 192 631 770 periods of the transition frequency between ground-state hyperfine levels of the 133Cs atom. Theory of such hyperfine structure depends in turn on the “fine structure constant”, R ) 2πe2/hc, for which the most precise determination is based on the g-factor for a single trapped electron. A more accurate value of R should make possible more accurate determination of various other fundamental constants of physics based on ultraprecise spectroscopic measurements of hyperfine transition frequencies. 3H-3He Mass Difference and the Neutrino Mass. Determination of the neutrino rest mass to a precision of (1 eV remains one of the most important experiments in modern physics, for several extremely fundamental reasons. First, if the neutrino were found to have a sufficiently large rest mass (say, g10 eV), then the huge number of neutrinos in the universe could provide some or all of the “missing mass” needed for gravity to pull the universe back together for another “big bang”. (That is reason enough.) Second, the experimentally observed flux of electron neutrinos from the sun is a small fraction (depending on the neutrino energy and detector) of what is computed by the best solar models. One possible explanation is that the observed electron neutrinos change (“oscillate” or “mix”) into the other two types (mu and tau) before reaching the earthsthat is possible, but only if the neutrino has mass. Third, the issue of whether the neutrino (like the photon) is its own antiparticle (Majorana particle) hinges on whether or not the neutrino is massive. The time spread in arrival of the neutrinos observed from the 1989 supernova explosion (since zero-mass neutrinos would all arrive together, independent of their kinetic energy) sets an upper limit of ∼15 eV on the neutrino rest mass.13 However, interpretation of that experiment is complicated by its indirect measurement of neutrino energy and the small number of observed neutrinos. Very recent attempts to detect neutrino “mixing” directly, by observing electron antineutrinos formed from muon antineutrinos, have been interpreted oppositely by members of the same research team.14 Therefore, the best current estimates for neutrino mass derive from careful analysis of the β-decay of tritium to helium-3, an electron and an electron-antineutrino:

H f 3He+ + e- + νje

3

(1)

The neutrino mass is determined from the “end point” of a Kurie plot based on measurements of the electron energy distribution from the β-decay process. The shape of the plot depends on both the neutrino mass and the 3H-3He mass difference (as well as the excitation energy of the 3He+ product and energy response function of the instrument). A recent such measurement yielded an upper limit of mνc2 ≈ 7 eV for the neutrino mass (expressed in energy-equivalent units).15 However, it is worth noting that the experiment actually gave a negatiVe (i.e., impossible) value of (mνc2)2 ≈ -39 eV2 for the square of the neutrino mass. The reported upper limit of a positiVe mνc2 ≈ 7 neutrino mass takes into account estimated statistical and

Mass Spectrometry systematic errors of 34 and 15 eV2. Thus, an independent mass spectrometric determination of the 3H-3He mass difference to a precision of better than 1 eV should significantly enhance the precision (and credibility) of the neutrino mass determined from β-decay experiments. Of course, mass spectrometry determines the mass difference between the ions rather than the neutral atoms, but the ionization energies are known to much higher accuracy than the mass difference being measured. The S.I. Kilogram. Although the atomic masses of the first 20 or so light elements are now known (relative to 12C) to about 1 part in 1010,8 macroscopic masses are known only to within about 10-100 ppm. The standard S.I. kilogram, dating from 1889, is still defined as the mass of a 90% platinum/10% iridium bar at the Bureau International de Poids et Mesures at Se´vres, near Paris. Unfortunately, even taking the standard bar out and cleaning it (which has happened only three times in this century) for comparison to the other 75-odd national prototype standards changes the mass of the object by parts in 108. However, since it is now possible to grow perfect single crystals of silicon weighing hundreds of grams, one can imagine redefining the kilogram to be a particular number of 28Si atoms in a silicon crystal. In fact, the limiting factor in such a definition is currently the precision with which the density of such a crystal is known (about 1 part in 107, which should improve to ∼1 part in 108 for a silicon crystal enriched in 28Si). Thus, it may soon be possible to use a silicon crystal with a known (say, from precise measurement of the crystal dimensions) number of atoms to monitor the current Pt/Ir standard bar and eventually to replace it. Proton vs Antiproton Mass: Test of CPT Invariance. One of the most important symmetry principles in physics, underlying the “standard model” which connects the various families of fundamental and composite particles, is the combined invariance of charge conjugation (C), parity (P), and time reversal (T). In other words, the outcome of any particle experiment is identical to a time-reversed mirror image of the experiment in which each particle is replaced by its antiparticle. Although C, CP, and T symmetries may be violated individually, it is not possible to construct a Lorentz invariant, local field theory which is not invariant under CPT.15 By use of an openended cylindrical Penning trap to determine the cyclotron frequencies of the proton and antiproton,16 Gabrielse et al. have recently shown that their mass-to-charge ratios are identical to within 1 part in 109. Although precision tests of CPT invariance have previously been performed for leptons and pions, the proton/antiproton mass-to-charge ratio comparison is more precise by a factor of 45 000 than other prior methods for baryons. Trapped Ion Mass Spectrometry Ion mass discrimination and detection embrace a large family of techniques based on related but different principles. In ionbeam instruments, ions of different mass-to-charge ratio are separated by electrostatic deflection, magnetic deflection, combined electrostatic/magnetic deflection, time of flight, ion mobility (in the presence of a collision gas), and/or massselective response to multipolar (especially quadrupolar) dc/ac electric excitation. For example, the middle diagram of Figure 117,18 shows isopotential surfaces for a two-dimensional quadrupole electric potential, which serves to guide (along the z-direction) a beam of ions of a wide or narrow range of massto-charge ratios, depending on whether the applied potential is ac-only or ac plus dc. Such quadrupole “mass filters” are the most common mass analyzers in use today. Paul and Penning Ion Traps. A qualitatively different approach is offered by ion “traps” (both Paul and Penning)

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Figure 1. Three electric isopotential surfaces. (left) three-dimensional axial quadrupolar potential, for ion confinement in a Paul or Penning trap; (middle) two-dimensional axial quadrupolar potential, for a quadrupole mass filter or ion guide for an ion beam, or axialization of ions in a Penning trap. (right) one-dimensional dipolar potential, for excitation or detection of trapped ions. Reproduced with permission from ref 17. Copyright 1989 Elsevier.

which have proved to be extraordinarily versatile tools for “chemical” mass spectrometry, because “parent” ions (and their reaction or photofragmentation “product” ions) may be confined for extended periods to allow for ion formation or injection, ion chemistry, ion fragmentation, and mass analysis. Unfortunately, it is impossible to confine ions in all three orthogonal directions by application of a static electric field alone, because of Laplace’s equation, ∇2Φ(x,y,z) ) 0, where Φ(x,y,z) is the electric potential field. For example, an attempt to confine ions in the z-direction by a simple harmonic potential, V(z) ∝ constant‚z2, requires a compensating potential in the x- and y-directions: V(x,y) ) -(constant/2)(x2 + y2), which produces a radially outward force (since force is the negative gradient of potential). The overall three-dimensional axial quadrupolar potential field is easily generated by applying fixed voltages to surfaces which match the isopotential surfaces of the leftmost diagram of Figure 1. Two clever approaches (leading to the Nobel Prize in Physics in 1989 for Dehmelt and Paul) for solving the problem of ion trapping both begin from the isopotentials of Figure 1. In the Penning trap, a static magnetic field applied along the positive or negative z-direction confines ions radially by virtue of their cyclotron orbital rotation about the magnetic field. In the Paul trap (also known as the “quadrupole ion trap”), the dc electrostatic potential is augmented by an additional ac electric potential; if the ac oscillation frequency is sufficiently high, then the combined potential may be regarded as an effectively static three-dimensional “pseudopotential” which rises quadratically in all three orthogonal directions away from the center of the trap. It is worth noting that the electric potential near the center of a trap of any shape approaches the three-dimensional axial quadrupolar limit; thus, Paul or Penning traps (“cells”) may take cylindrical or tetragonal forms.19 Ion Manipulations. The breakthroughs which vaulted Paul and Penning traps to the forefront of mass spectrometry derive from new methods for excitation and detection of ion “normal mode” motions (i.e., axial and radial “secular” motions in the Paul trap;20,21 cyclotron rotation, magnetron rotation, and axial oscillation in the Penning trap16). In the Paul trap, ions may be ejected axially (to a detector) by “mass-selective instability”, i.e., by scanning the amplitude of the ac voltage at fixed dc voltage amplitude.22 Alternatively, ions in a Paul trap may be heated translationally by resonant excitation at the axial “secular” frequency, to produce collisionally activated dissociation (CAD), also known as collisionally induced dissociation (CID), followed by a mass-selective instability scan to produce a “tandem” or “MS/MS” experiment.23 In the Penning trap, ions of a given mass-to-charge ratio are resonantly excited at their cyclotron frequency, either to increase ion translational energy for CID MS/MS or to drive the ions to a larger cyclotron

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Bowers et al. analysis of complex mixtures (e.g., crude oil distillates,6 fullerenes,43 and synthetic44,45 or biological46,47 polymers), as well as for detailing ion-molecule reaction mechanisms and ion structure.38,48-51 A number of these applications will be amplified in later sections of this article.

Figure 2. Time-domain ICR excitation wave forms (left) and their corresponding frequency-domain FT magnitude-mode spectra (right). Proceeding from top to bottom: rectangular (“top hat”), frequencysweep (chirp), broad-band SWIFT, and windowed SWIFT (e.g., for broad-band excitation of species of all but a narrow range of frequencies).

orbital radius for subsequent detection of the image charge induced on one or more electrodes; Fourier transformation of the time-domain image charge signal yields the whole mass spectrum at once.24,25 Evolution of many Paul ion trap applications to ion structure and ion-molecule reaction chemistry, kinetics, equilibria, and energetics has followed prior parallel developments in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, much as many FT-ICR techniques themselves followed prior developments in Fourier transform nuclear magnetic resonance spectroscopy.26 A good example is the use of stored wave form inverse Fourier transform (“SWIFT”) excitation, in which any desired frequency-domain excitation spectrum is generated by inverse Fourier transformation (and suitable phase encodement) to yield a time-domain stored wave form which may be sent to Paul trap side electrodes or Paul trap end-cap electrodes to increase the kinetic energy of ions of arbitrary mass-to-charge ratio range(s).27,28 Figure 2 shows that the frequency-domain profiles resulting from simple “top-hat” or frequency-sweep “chirp” excitation are broad-band, but neither uniform in magnitude nor sharply selective in frequency. SWIFT excitation, on the other hand, produces optimally uniform-amplitude frequency-selective excitation and is rapidly becoming the method of choice for isolating trapped ions for subsequent structural or reactivity studies. Ion Trap Applications: a Sampler. The impact of trappedion (both quadrupole ion trap and ICR) mass spectrometry may be gauged in part from the numerous books,20,29-32 journal special issues,33-35 and 50-odd reviews on the subject over the past half-dozen years or so, a few of which are listed here.18,19,21,36-40 Both the Paul trap and FT-ICR instruments can approach attomole detection limits, either by destructive detection of individual ions (Paul trap) or by nondestructive repeated remeasurement of the same ions (FT-ICR).41,42 Thousands of Paul traps are in use as gas chromatograph (and more recently, liquid chromatograph) detectors, and Paul traps have been widely applied to low-resolution MS and MS/MS analytical, chemical, and biological problems. FT-ICR offers the highest mass resolving power and mass accuracy of any mass analyzer, with almost 200 high-resolution instruments in use worldwide. FT-ICR offers unsurpassed analytical value for

Special Mass Spectrometry (MS) Techniques Separation/MS. The sensitivity and speed of MS make it an ideal complement to separation methods; instruments combining gas chromatography on line with MS (GC/MS) have accounted for the great majority of MS sales for two decades.52 GC/MS is ideal for complex mixture identification, such as drug metabolites, comatose patient body fluids,53 forensic samples, pollution control, and process monitoring. A 1 h GC/MS measurement can produce 100 or more electron ionization (EI) mass spectra; these can be matched on-line for identification against the current reference database of 275 000 different EI spectra, each in 1 s on average.54 Newer such “combination” techniques couple methods such as liquid chromatography (LC) and capillary electrophoresis with MS, so that nonvolatile samples can be analyzed with appropriate ionization techniques (vide infra). More complex samples can be analyzed with improved mass spectrometry; for crude oil samples, the >105 resolving power of FT-ICR MS can separate many hundreds of molecular ions, such as the isobars C19H14S (274.0816), C16H18S2 (274.0850), C20H18O (274.1358), C21H22 (274.1722), C18H26S (274.1755), and C20H34 (274.2660).6 Tandem Mass Spectrometry.55 The mass spectrometer is basically a separation device; this capability can be coupled to an identification MS to yield the far faster MS/MS combination method for mixture analysis. A “soft” ionization of the mixture produces molecular ions with masses indicative of the mixture components; mass separation and fragmentation of one of these gives product ions whose mass spectrum is characteristic of that component’s structure. Further, the structures of smaller product ions, such as CH3CHdOH+ vs CH3O+dCH2, can be distinguished by comparison of their spectra produced by high-energy (4-10 kV) collisionally activated dissociation against those of reference isomeric ions.56 By far the greatest use of the triple quadrupole and other tandem mass spectrometers has been for the analysis of “targeted compounds” in complex mixtures to achieve much lower detection limits (reduced “chemical noise”) and higher specificity.55 Even if the mixture contains several components that produce molecular ion masses of that of the desired component, its MS/MS dissociation will almost always produce abundant fragment masses different than those of the other molecular ions of its molecular weight. For example, quantitative analysis of individual drug metabolites in blood extracts is possible at the 10-12 g level using internal standards labeled with stable isotopes.55 Separation/MS/MS. Combining GC or LC with MS/MS also provides a powerful analytical tool of unique specificity and sensitivity. An unusually impressive example involves the structural characterization of the individual nonapeptides presented by a virus-containing cell’s major histocompatability complex that is recognized by a killer T cell.57 The sample, isolated from liters of cell culture, contained thousands of different oligopeptides, but the active nonapeptides were only present at the subpicomole level. The electrospray ionization (ESI) mass spectra of the biologically active fractions separated by capillary LC showed several molecular ions. MS/MS of these gave nonapeptide sequences; synthesis then provided definitive proof of biological activity. LC/MS/MS now is also a powerful tool for protein sequencing.58 The protein is chemically or enzymatically digested to

Mass Spectrometry produce a complex mixture of oligopeptides. These are then separated by LC, with on-line MS monitoring showing that most fractions contain a number of different molecular ions. The MS/MS spectrum from each of these can provide its sequence, or at least an extensive characterization.59 Overlapping these oligopeptide sequences can then provide information on the protein sequence, data that have been especially valuable for verifying protein sequences obtained by classical means. MSn Ion Characterization. LC/MS/MS sequencing of the 4-10 kV ions of magnetic sector instruments can be effective for singly charged ions as large as 3 kDa.58 This upper limit is reduced to 10-6 s) H2Cl neutrals,69 a commonly proposed intermediate in the longstudied H2 + Cl2 reaction that had not been observable previously by conventional experimentation. Mass Spectrometry of Large Molecules Ionization Techniques. Surely one of the most dramatic recent improvements in mass spectrometry has been the capability for structural characterization of biomolecules and polymers. Twenty years ago the mass spectra of molecules larger than molecular weight 600 were rarely measured, with only field desorption70 and 252Cf-plasma desorption71 showing that larger molecules could be ionized by special techniques. A decade ago fast atom bombardment (FAB)72 became a popular ionization method for somewhat larger molecules, but a 6 kDa application was still rare. Now, however, detailed structural information is being obtained from 60 kDa molecules, with isolated reports of the detection of those larger, even 100 MDa plasmid DNA.5 Both matrix-assisted laser desorption ionization (MALDI)2 and electrospray ionization (ESI)4 have now emerged as routine techniques available from almost all MS instrument manufacturers, used in a thousand or more laboratories. In MALDI, the sample molecules are dispersed in a lowvolatility solid matrix that is flash vaporized by a high-intensity laser pulse of photons absorbed by the matrix but not by the sample (e.g., 355 nm). In contrast, for ESI a dilute aqueous solution of the sample is sprayed from a capillary through a

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Figure 3. MALDI/TOFMS mass spectrum of lipoproteins (6-29 kDa) obtained from a delipidated high-density lipoprotein fraction of human plasma. Reproduced with permission from ref 2.

several kiloelectronvolt potential; evaporation of the electrostatically charged droplets leaves intact sample molecules in the gas phase containing a multiplicity of charges. Thus, peaks in an ESI spectrum usually appear between m/z 600 and 2000 regardless of the mass of the neutral species, a great advantage for limited m/z range instruments. This orders-of-magnitude increase in the molecular size capabilities of MS has provided a high incentive for developing corresponding increases in instrument capabilities, particularly for mass range, resolving power, multichannel detection, sensitivity, and tandem mass spectrometry (MSn). Just as the new capabilities of FAB with MSn provided a driving force for the development of improved tandem double-focusing magnetic sector73 and hybrid sector-quadrupole55 instruments, MALDI2 inspired the rediscovery of the time-of-flight (TOF) instrument. It provides virtually unlimited mass range and measures all ion masses in 105 at m/z 1000 is of critical value in several ways. Isotopic peaks separated by 1 Da (∆m ) 1.0034 Da for 13C vs 12C) can now be resolved for the 86 kDa dimer of creatine kinase (Figure 4).78 The presence of 1.1% of 13C in 12C, and of other minor isotopes such as 2H, 15N, 18O, and 34S, makes the mass of the most abundant isotopic peak of this dimer 54 Da higher than that of the monoisotopic peak. Although only the m/z value of a peak is measured, the 1 Da spacing of its neighboring isotopic peaks provides a convenient scale for extracting the accurate m value.79 Conventional deconvolution methods for low-resolution ESI spectra are ineffective for large molecule ESI spectra containing many mass values.80 Sample and Ion Purity. A stringent requirement for classical methods of protein and DNA sequencing, and for more sophisticated instrumental methods such as NMR and X-ray crystallography, is that the samples examined should be pure. A unique advantage of MSn is that purity is not required. ESI “soft” ionization (producing molecular ions without significant fragmentation) of a mixture indicates each sample component by its corresponding molecular ion, whose structure can then be characterized by the ion’s isolation, dissociation, and fragment ion mass analysis. Further steps of MSn can provide additional characterization for larger fragment ions. However, ion “purity” is also important. Both MALDI and ESI can cause fragmentation as well as ionization, and these product ions could be confused for impurity molecular ions. In being unusually “soft”, the ESI ion internal energies are often too small to break up noncovalent associations of molecular ions with impurities or solvent molecules. Infrared irradiation of the trapped ions64 is promising in removing noncovalent adducts from both positive protein ions (e.g., albumin, 67 kDa)80 and negative nucleotide ions (e.g. a 100-mer DNA)81 formed by ESI, while causing minimum dissociation of covalent bonds in the molecule. However, a very promising ESI application uses the relative binding energies of such noncovalent complexes in the gas phase to predict those in solution, such as the binding

Bowers et al.

Figure 5. ESI/FTMS mass spectrum of purified recombinant thiaminase I. (top) The 39+ molecular ion region (20 scans). (bottom) The 38+ region after incubation of thiaminase I with the 4-amino-6chloro-2-methylpyrimidine (10 scans).

of the immunosuppresive drugs FK506 and rapamycin to the cytoplasmic receptor FKBP.82 Adduction by cations or anions can also produce “impurity ions”. Cations such as Na and K are common impurities in both positive and negative ion mass spectra.4 For example, the phosphate backbone of nucleotides, for which negative ion mass spectra are the most favorable, have a high tendency to retain alkali ions. The ESI mass spectrum of a 76-mer transfer RNA showed up to 50 adducted Na atoms per molecule without prior salt removal from the sample.81 Positive protein ions can exhibit adducted anions such as phosphate or sulfate.83 Sample cleanup alone is no guarantee of adduct removal, and for an unknown sample the dissociative tendency to lose water or similar small molecules on ionization is also difficult to predict. For a 50 kDa molecular ion, the presence of a Na adduct (+22 Da) or H2O-loss ion (-18 Da) would not be separable with RP ) 2000. In fact, because of the overlap of the multitude of isotopic peaks, these impurity ions would not be separated without isotopic peak resolution (RP ) 50 000). Obviously, the mass centroid value of the peak containing such overlapping mass values would be in error, a problem that is nicely solved by the FT-ICR advantage of RP > 105, which allows impurities to be distinguished and identified.47 The high-resolution capability of FT-ICR is also critical for correctly identifying the ion charge multiplicity that forms most fragment ions of a 50 kDa protein or nucleotide between m/z 600 and 2000. In Figure 4, molecular ions are observed with 20-46 charges for ESI of recombinant creatine kinase from rabbit muscle (43 kDa).77 The FT-ICR spectrum of a 23 kDa poly(ethylene glycol) shows ∼5000 separated isotopic and charge state peaks of 48 oligomers.84 With FT-ICR, the desired precursor ion for MS/MS can be selected from those trapped in the ion cell using either SWIFT26,27 or quadrupolar axialization.41,85 Selection of a single isotopic peak of a 29 kDa protein ion for MS/MS has been achieved with RP ) ∼20 000.86 Molecular Weight (Mr) Determination. Conventional separations of macromolecules, such as SDS-PAGE, give only approximate indications of the Mr value. The far higher accuracy of mass spectrometry can provide much more useful Mr values to verify other structural assignments and identify post-translational modifications, heterogeneity, and active sites. MALDI is especially convenient in that commercial instruments now are available for routine use by research scientists with no

Mass Spectrometry

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Figure 6. Dissociation spectral data (fragment mass values for 13Cn peak, n value italicized) of rabbit muscle CK, correlated with the cDNA sequence and Pro positions (heavy lines, cleavages N-terminal to Pro). Horizontal boxes on the left and right represent b-type and y-type fragments, respectively (those in between are internal fragments), with the numeral representing the number of amino acid residues in the fragment. External columns: expected mass differences for indicated amino acids. Inset: isotopic distributions of key fragment ions per Figure 4 and legend. Reproduced with permission from ref 78.

special training. These can provide Mr values in minutes with accuracies of 0.01-0.1% in cases where there are no interfering impurities or adducts not distinguished by resolving power limitations. Illustrating the advantages of ESI/FT-ICR, the Figure 4 spectrum of creatine kinase shows Mr ) 42 982-27 (actually the mass of the most abundant isotopic peak; the last digit in italics gives its additional mass relative to the mass of the monoisotopic peak).78 This value is within 1 Da of the Mr value from cDNA sequencing, providing an independent verification of the sequence data. More importantly, isoelectric focusing separation has shown that the creatine kinases are mixtures, with this sample containing at least three components. The Figure 4 spectrum shows that all components have the same molecular weight (2 Da, contrary to proposed explanations such as methylation or other post-translational modifications, but consistent with one or more steps of deamidation (-NH2 f -OH, 16 f 17 Da).87 As a contrasting example, the ESI/FTICR spectrum of 42 kDa thiaminase (Figure 5)88 shows three distinct molecular ions separated by 71 and 57 Da, the separation expected for the additional amino acids alanine and glycine. Reaction with a pyrimidine that mimics the reaction of thiamin with the enzyme adds the expected 108 Da to each of these molecular ions, indicating that each contains the active site.78 ESI/FT-ICR of other large proteins indicates that erroneous Mr values derived from classical sequencing are not uncommon. For example, the published sequence of porcine albumin yields Mr ) 66 740, but the ESI/FT-ICR spectra of two different samples give values of 66 736 and 66 886, with the spectrum

of their mixture confirming this mass difference.80 The initial ESI spectra (RP ) 160 000) showed unit-mass-separated isotopic peaks over a range of >1000 Da; these represented noncovalent adducts, as gentle IRMPD of the gaseous ions removed the adducts to give the observed Mr values. Accurate molecular weights can also be obtained for larger nucleotides. For example, the ESI/FT-ICR negative ion mass spectrum of phenylalanine yeast transfer-RNA shows Mr ) 24 950.3 vs 24 950.5 expected. For a 100-mer DNA, again requiring IRMPD adduct removal, a value of Mr ) 30 702.4 was found vs 30 702.1 expected.81 MSn Sequence Information. Dissociation of protein or nucleotide ions provides direct information on the ordering of their naturally occurring amino acids (19 unique masses) or bases (4 unique masses). CAD and IRMPD dissociations effect mainly cleavage of the amide bonds, with products designated as b- and y-ions for the N- and C-terminal products, respectively. Further dissociation of a b- or y-product is also observed to produce a smaller ion containing the same terminus plus an internal (i) product ion.89,90 Several methods for distinguishing b- and y-ions have been proposed.90 Dissociation of the molecular ions of creatine kinase (Figure 4) yielded over 70 fragment ions identified directly in a single spectrum (Figure 6)78 representing 53 fragment mass values. (Some represent the average of two or more charge states.) For thiaminase (Figure 5), none of the molecular ions had masses corresponding to the DNA-determined sequence; MS/MS gave fragment ions that restricted the location of the error and of the active site.88

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Figure 7. ESI/FTMS sequence data of 50-mer DNA treated as an unknown. (top) Infared multiphoton dissociation (IRMPD) of molecular ions. (middle) Internal series from IRMPD. (bottom left) Nozzle-skimmer dissociation. These data provide the complete, correct sequence. Reproduced with permission from ref 92.

For a linear multiply charged biomolecular ion, dissociation of a single bond can create a pair of products of complementary mass values.89 Such products from chemical or enzymatic degradation of such large biomolecules are relatively rare. Classifying these as N- or C-terminus ions then makes possible a “top-down” approach to biomolecule sequencing,96 in which such complementary ions can provide a skeleton for mapping the fragment ion locations into the whole molecule. For the creatine kinase example,78 the mass sum of the Figure 6 fragment ions 24 087.21-15 + 18 895.27-12 ) 42 982.48-27 compares very favorably with the molecular ion measured mass of 42 982.24-27. Note that dissociation of the 18 895 Da fragment ion, y168 (yn, n indicating the number of amino acids) produces y98 + i70 and y48 + i120. As utilized for oligopeptide sequencing,58 mass differences can now identify individual amino acids in the sequence, as indicated in the outside columns of Figure 6. Identification of the 10-residue sequence from b52 to b62 (average mass error only 0.03 Da) is more than sufficient for the design of an oligonucleotide probe.91 DNA Sequencing. ESI-produced DNA negative ions dissociate much more readily than positive protein ions, so that these nucleotide fragmentations show extensive sequence information. Despite the correspondingly high spectral complexity, the complete sequence of a 50-mer can be derived from MS2 and MS3 spectra using ESI/FT-ICR (Figure 7).92 The

fragment mass values are much more definitive of sequence than those in protein mass spectra, as there are only four possible bases. Again, sequencing used the “top-down” approach90 of identifying complementary ion pairs and triplets plus individual base assignments from mass differences. An unknown 50-mer, synthesized to contain only one base substitution, was correctly identified as a 27A f 27T mutation from a ∼1 min experimental measurement.92 Gaseous Protein Conformations. Trapped ion instruments afford the opportunity for ion characterization by derivatization.51,66 For larger multiply charged ions of cyctochrome c in the FT-ICR cell, exchange with D2O occurs at six or more discrete levels dependent on ion preparation and charge state, representing exchange at 30%-70% of the sites effective in solution. Presumably, the gaseous ion conformation restricts the number of sites accessible in the gas phase. IR laser heating “unfolds” the ion to make more sites available, while charge stripping can fold as well as unfold ion conformations.93 In solution the N- and C-terminal R-helices exhibit the highest free energy of unfolding, but CAD of the deuteriated species shows these to exchange readily in the gas-phase ions. Metastable Ions The initial focus in mass spectrometry was simply measuring mass (or more accurately mass-to-charge ratios), and all early

Mass Spectrometry

J. Phys. Chem., Vol. 100, No. 31, 1996 12905

a

b Figure 8. Hypothetical potential energy surfaces for the metastable reaction of nascent (MA)+ collision complexes formed from the reaction of a metal ion M+ with a neutral molecule A. In type I complexes there is no reverse activation barrier for product formation (MB+ + C) while in type II complexes a significant barrier exists. The expected forms of the kinetic energy release distributions are given on the righthand side of the figure. Note that Emax is always less than the available energy, ∆Hrx. For type II surfaces the dotted line exemplifies the usual KERD observed.

designs simply did that.94-97 However, the need for higher resolution ultimately led to coupling magnetic and electric sectors, the most important being the Nier-Johnson design,98 in which an electrostatic sector (ESA) was followed by a magnetic sector and the ions “double focused” (i.e., spatial and velocity focusing). One of the benefits of double focusing was its ability to greatly reduce unwanted “metastable ions”, ions that dissociated after acceleration but before magnetic analysis in single-sector machines. These ions appeared in some of the early mass spectra99 but were not explained until the mid1940s.100 An excellent monograph has been written that discusses the origin of metastable ions and methods developed to study them up to 1972.101 In a two-sector instrument, metastable ion reactions can occur in the field-free region either just before the first sector or between sector 1 and sector 2.

M1+* f M2+ + M3

(2)

Early work was done on Nier-Johnson machines. First, fieldfree region metastables could not normally be detected since the reaction products had less mass, and hence less kinetic energy, than the M1+ parent ions. The problem was overcome by development of the so-called V-scan102 where the acceleration voltage V was increased to exactly offset the energy lost due to the loss of mass and allow transmission of M2+ through the ESA. Often the resulting M2+ peaks were broad due to kinetic energy released in the fragmentation process,103,104 and approximate methods were developed to extract this information.101 It was, in fact, this energy release aspect of metastable reactions that caught the attention of physical chemists since such studies revealed information about the potential energy surfaces governing unimolecular dissociations. A major advance occurred with the development of so-called “reverse geometry” machines where the magnetic sector preceded the ESA.105 In this case metastable reactions of mass-

Figure 9. Kinetic energy release distributions for metastable dissociation of nascent Co+ (isobutane) collision complexes: (a) loss of H2 to form Co+ (isobutene) and (b) loss of CH4 to form Co+ (propene). Both experimental and statistical phase space theory models are shown. Reproduced with permission from ref 112.

selected ions were routinely performed by scanning the ESA and utilizing the relationship M2+ ) M1+ (E2/E1). The resulting scan of E yielded a peak shape exactly centered on the value of E2. If instrument discrimination could be eliminated,106 this peak shape could be analyzed to yield the M2+ kinetic energy release distribution (KERD).107-109 The study of metastable ions has contributed across the chemical landscape, yielding information on ion structures, energetics, and dissociation mechanisms.101 Here, however, we will focus on only one aspect of metastable dissociations, the generation and interpretation of KERDs. There are two generic kinds of potential energy surfaces (PES) that yield characteristic KERDs. These are shown schematically in Figure 8. The type I surface has no reverse activation barrier, and a “statistical” KERD consistent with phase space theory calculations110 is expected. These KERDs peak at low energy and have maximum releases substantially less than the available energy (∆Hrx in Figure 8). On the other hand, when a reverse activation barrier occurs in the exit channel along the reaction coordinate (type II surface), a broad KERD is expected that peaks well away from zero. If the repulsive surface is especially steep, resulting in a rapid dissociation and little intramolecular energy transfer, a shift in the onset away from zero can also occur, although such instances are rarely observed. A reaction that nicely exemplifies both type I and type II surfaces is the dissociation of metastable Co+ (isobutane)

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nascent collision complexes:112 Co+ + i-C4H10

[Co+(i-C4H10)]*

(3)

Co+(C

(4a)

4H8)

+ H2

[Co+(i-C4H10)]* Co+(C3H6) + CH4

(4b)

The KERDs are given in Figure 9a,b, along with predictions of statistical phase space theory. The KERD for the CH4 loss channel peaks near zero and smoothly falls off at high energy and is exactly fit by phase space theory. On the other hand, the H2 loss channel has a complex of KERD with a high-energy bump that is clearly poorly fit by phase space theory. In fact, the KERD is suggestive of two mechanisms for H2 loss: one possibly statistical and one clearly with a reverse activation barrier. Similar complex KERDs have been observed for H2 loss from M+(propane) complexes113,114 for M ) Fe, Co, and Ni and have been shown to result from initial metal insertion into different C-H bonds (primary, secondary, etc.). The excellent fit between theory and experiment in the CH4 loss channel was obtained by using ∆Hrx as a free parameter in the phase space simulation. The theoretical KERD is extremely sensitive to this parameter, and consequently an accurate determination of the Co+-propane bond energy (44 ( 5 kcal/ mol) was obtained.112 An identical value of this bond energy was obtained by fitting the KERD of the C2H4 loss channel from decomposition of nascent Co+ (c-C5H10) complexes.112 The method has proven reliable and has generated a number of bond dissociation energies between metals and simple ligands that are difficult to obtain by other means. There have been literally thousands of metastable reactions studied, and for the most part they fit either the type I or type II surfaces of Figure 8. However, occasionally a reaction occurs that apparently fits neither case and presents an opportunity to learn something new. Such a circumstance occurred in the metastable reaction of nascent H5+ clusters: +

+

H3 + H2 f [H5 ]*

(5a)

[H5+]* f H3+ + H2

(5b)

The [H5+]* intermediate in this deceptively simple reaction is expected to have a lifetime of ∼10-9 s according to statistical theory calculations. The reaction should definitely occur on a type I surface since the nascent [H5+]* complex is formed on essentially every collision via reaction 5a, and hence there cannot be a barrier along the reaction coordinate. Nonetheless, metastable formation of H3+ from H5+ was experimentally observed.115 Further, when very careful ultrahigh-energy resolution experiments were done, a highly structured KERD was obtained as shown in Figure 10. Similar structure was observed in loss of D2 from [D5+]*112 and H2 from [H6+]*116, but few if any other examples have been reported. The eventual explanation entailed tunneling of H2 from quasi-bound vibrational states existing behind a series of centrifugal barriers for bound J states of the many H5+ geometric isomers. By building on ab initio surfaces117 and a theoretical model118 developed for H atom tunneling in CH4+ 119 and NH3+,120 the theoretical KERD shown in Figure 10 was obtained. The good agreement with experiment confirmed the basic assumptions in the approach. This is a perfect example of how the simplest systems often provide the most interesting case studies. Bond Energies Mass spectrometry has played the central, nearly exclusive role in generating accurate information on ion energetics. Brief

Figure 10. Kinetic energy release distribution for the metastable decay of nascent (H3+-H2)* collision complexes. The theoretical KERD was obtained from a model of H2 tunneling from quasi-bound vibrational states of (H5+)* through rotational barriers of the various allowed J-states of the system (see text). Reproduced with permission from ref 115.

mention was made in the section on Metastable Ions about obtaining such information from analysis of KERDS. Much more extensive information on transition metal-ligand bond energies has come from ion-beam studies121 as discussed elsewhere in this issue.9 A recent compilation of these and other M+-ligand bond energies has been assembled.122 The most accurate and most extensive studies entail measurement of ionic equilibria, of which the two prototypes are

AX+ + B a BX+ + A

(6)

AL+n-1 + L a ALn+

(7)

Measurements of equilibrium constants and their temperature dependence allow determination of thermodynamic quantities using the relationships

∆G°T ) -RT ln Keq

(8)

∆G°T ) ∆H°T - T∆S°T

(9)

In many cases, T ) 0 K values can be accurately estimated using the methods of statistical mechanics to extrapolate data taken at experimentally accessible temperatures. Clustering equilbria studies (reaction 7), using high-pressure mass spectrometry, were initiated in the 1960s. Many very important experiments were carried out early on, including the fundamentally important sequential hydration of H3O+ 123 and the hydration of the entire series of halide ions124 and alkali ions.125 A vast number of binding energies have subsequently been determined and compilations published.126 Conformation of the accuracy of these results by other methods has been limited, although addition of the first rare-gas ligand to transition metal ions has been studied using both equilibrium127 and spectroscopic methods128 with excellent agreement. A more detailed discussion of clustering and solvation is given elsewhere in this issue.129 The prototypical ligand exchange equilibrium, reaction 6, involves proton transfer130,131 and yields very accurate values of gas-phase basicities, ∆G°AB. Since clustering is not involved, entropy terms are usually small and can be accurately estimated from statistical arguments yielding reliable values of relative proton affinities, ∆H°AB. By using compounds with similar

Mass Spectrometry proton affinities, self consistent sets of data can be obtained leading to accuracies in ∆H°AB of the order 0.1 kcal/mol (0.004 eV or 35 cm-1). Never before had an intrinsic property like “basicity” been measured to this accuracy. This led to a major rethinking of how molecular properties affected basicity as well as the conjugate property acidity.132 What became quickly apparent was the traditional, solution-based, ordering of the basicity and acidity of molecules was strongly dependent on solvation effects133-135 and bore little resemblance to the ordering of the intrinsic gas-phase properties. Compilations of the early work in the area exist.132,136,137 Since equilibria measurements only yield relative basicities or acidities, the relative “ladder” must be tied to an absolute measurement at at least one point, and preferable several points, in order to obtain absolute values. While this caused some problems early on, both accurate relative and absolute values now exist.138,139 Ion Structure Successful determination of molecular structure is one of the great success stories of modern physical chemistry. However, the elegant methods developed for stable neutral species, and even reactive species like radicals, are far too insensitive to apply directly to molecular ions. Hence, clever and often indirect methods must be employed to obtain structural information in almost all instances (although some “nearly” traditional methods have been applied to a number of small molecular ions140,141). One exotic method is coulomb explosion.142 In this experiment small ions are accelerated to megaelectronvolt energies and impacted on a thin foil. The initial interaction with the foil strips off most of the electrons of the particle. The bare nuclei then coulomb explode and are detected in coincidence on a spatially resolved analyzer plate. While the data analysis is not straightforward and controversy still exists, the method has been successfully applied to a number of small hydrocarbon ions. For example, it has been shown that C2H3+ is basically a linear H-CdC-H moiety with the third hydrogen bridging the two carbon atoms,143 a result consistent with high-level theoretical calculations.142 Similarly, C3+ appears to have a triangular carbon structure while in C3H4+ the carbon atoms form a linear substructure.142 The results for C3H3+ appear to indicate both isomers are present, in approximately 70% (cyclic) and 30% (linear) abundances.142 The presence of both cyclic and linear isomers is consistent with ICR,145 SIFT,146 and CAD147 results. Theory148 indicates c-C3H3+ is 60 kcal/mol more stable than the linear isomer, and hence observation of varying proportions of linear C3H3+ isomer from different precursors147 indicates kinetics dominates the formation mechanism (electron impact on “linear” allene or methylcyclopropane) rather than energetics. An indirect method for structural determination entails the use of ion-molecule reactions.51,66 Initially this method was used to distinguish between isomeric ions. For example, protonated oxirane eliminates H2O when reacted with PH3, but protonated acid aldehyde does not.149 This reaction proved useful because it was difficult to distinguish these two species using CID/CAD methods. Recently, a novel use of ionmolecule reactions has been developed. In these studies it was noted that distonic ions, where the charge and radical site in the molecule are separated, often undergo reactions quite different from their traditional radical cation isomers. One reagent that proved especially useful in this regard is dimethyl disulfide.150 Traditional radical cations usually undergo rapid electron transfer with this reagent, while distonic ions most often abstract a CH3S• moiety (sometimes accompanied by subsequent

J. Phys. Chem., Vol. 100, No. 31, 1996 12907 charge exchange) allowing unambiguous determination of reagent ion structure. The dominant method for ionic structure determination is CID/CAD56 as previously discussed. While indirect, this method can yield useful qualitative structural information. The structural information obtained is almost always of the “connectivity” variety, indicating the order in which the atoms are connected but offering essentially no direct information on conformation. Conformational information can be obtained using a relatively new development of an old technique: mobility measurements of target ions drifting through He gas. These kinds of experiments have been around for a long time151 and have even resulted in the development of an analytical device.152 However, it is the recent development of the ion chromatography (IC) method153 that allows accurate structural and conformational information to be obtained on a wide range of molecular ions. The critical new aspects of the IC method are formation of ions external to the drift cell (where mobility occurs) and sophisticated theoretical analysis that allows identification of ionic conformation from accurate mobility measurements. The first new aspect allows a large variety of sources to be coupled to the mobility cell including laser desorption,154,155 MALDI,156 ESI,157 EI,158 and SI.159 Ions emanating from these sources are mass selected before injection into the mobility cell, eliminating ambiguity in the species being studied. While the range of systems being investigated is rapidly expanding, it is in the area of carbon clusters153,154,160 that the method established itself as a unique and important contributor in the field of gas-phase ion structure. For example, for C36 the five structures given in Figure 11 have been unambiguously identified.154,161,162 By following the development of such structures from very small sizes to above C60 for both cations154 and anions162 and by performing critical annealing experiments,163 the first real evidence on how fullerenes are made in carbon arcs has emerged.164,165 Recent applications of the method have been on larger systems such as polymers,156 host/guest pairs,166 and large biological molecules.157 A second, distinct application of IC has also been developed158 that deals with electronic rather than geometric structure. It was noticed early on167 that transition metal ions with different electron configurations (e.g., 3dn or 4s13dn-1) could easily be separated using IC, and in favorable cases, individual electronic states within an electronic configuration could be distinguished. An example is given in Figure 12 for V+ ions generated by EI on VOCl3:168

VOCl3 + e– (200 eV)

V+ + (OCl3) + 2e–

(10a)

VCl+ + (OCl2) + 2e–

(10b)

VO+ + (Cl3) + 2e–

(10c)

where the eliminated neutral species are collected in parentheses since their identity is not determined in the experiment. The top trace in Figure 12 results from V+ ions directly generated by EI (reaction 10a) and injected into the drift cell containing 5 Torr of He. By observing changes in this arrival time distribution with electron energy and by comparing it to other first-row elements, assignments of the observed features could be made.158 The small single peak at long times (17% of the total V+ intensity) is due to V+ ions in the 3d4 configuration and contains the 5D ground state. The partially resolved doublet at short times is composed of the lowest two excited states of 4s13d3 configurations. The component at shortest times is the 3F second excited state (at 1.104 eV) and the longer time

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Bowers et al. slowly to the 5D ground state.168 He

He

V+(3d34s1;3F) 9 8 V+(3d34s1;5F) 9 8 V+(3d4;5D) fast slow

(12)

This is a surprising observation since the initial fast step has an energy deficit of 0.74 eV and the slow step an energy deficit of only 0.34 eV. Factors governing such processes are yet to be determined. In summary, the IC method offers a unique opportunity to probe transition metal state distributions, their formation mechanisms, deactivation rates, and state specific reaction rates and consequently holds much promise in further investigations of these important species. Summary and Prognosis Figure 11. Structures of stable C36 isomers that have been identified in both cationic and anionic carbon clusters (see text).

Figure 12. Arrival time distribution for V+ ions formed three separate ways. In the top trace, V+ is formed by 200 eV EI on VOCl3. In the middle trace, V+ is formed by injecting the EI generated fragment VCl+ into the IC cell at 50 eV. The bottom trace results from injecting VO+ at 150 eV. The single peak at long times contains the 3d4 5D ground state, and the doublet at short times correspond to the 3d34s1 3F and 5F excited states (see text).

component the 5F first excited state (at 0.363 eV). From the splitting in the peaks it appears that spin coupling contracts the s-orbital radius in the 3F state ∼2% relative to the 5F state. A second feature is observed in the bottom two traces of Figure 12. In this case the V+ ions are made by injecting either VO+ or VCl+ at high energy into the IC cell and collisionally dissociating them:

VX+ (150 eV) + nHe f V+ + X + nHe

(11)

where X ) O, Cl. In this case the ground state ions dominate (92%), and the excited state ions are formed exclusively in the lower lying 5F excited state. Hence, if the VO+ or VCl+ ions are formed electronically excited, this excitation is efficiently removed in the violent collision process. The V+ product ion state distribution is characteristic of a dissociation product “temperature” of ∼1600 K for both injected species. Finally, the 3F state collisionally deactivates relatively efficiently to the 5F state, but the 5F state deactivates much more

There are several ways to judge the current and future impact of mass spectrometry. To begin with, more than 50 000 mass spectrometers have been installed worldwide, and they are being replaced and augmented at a current annual sales volume of ∼$1 billion/year. As another indicator, 5 years after it began publication, a research journal devoted exclusively to mass spectrometry has risen to become the number three-ranked spectroscopy journal in the world. In terms of growth, the number of publications in mass spectrometry has held to a steady doubling time of about 6 years. More fundamentally, the best science changes people’s thinking. In that respect, mass spectrometry has assumed an increasingly important role, radiating from a physical chemistry center. For example, mass spectrometry opened up a whole field of cluster ion structure and chemistry. Mass spectrometry has produced and characterized experimentally a host of other species that were previously observed only spectroscopically in interstellar space. Mass spectrometry is beginning to provide the experimental data that will make it possible to test theories about the role of solvent water on protein structure and folding, and well as noncovalent binding between macromolecules and the whole concept of “hydrophobicity”. Mass spectrometry is making possible detailed chemical analysis at the femtomoleto-attomole level, leading to functional chemical analysis of living brain tissue to identification of immunologically determinant differences between tumor and normal cells. Mass spectrometry is providing the most precise tests of several fundamental questions in physics such as measurements of the neutrino mass, proton/antiproton mass ratio, etc. In summary, mass spectrometry is providing tools that allow researchers to attempt problems previously thought unassailable across a broad range of science, from nuclear physics to biology. With the explosion over the past decade in upper mass limit (from ∼2000 to more than 1 000 000 Da), mass-resolving power (from ∼100 000 to ∼1 000 000 at m/z up to 2000) and corresponding mass accuracy, sensitivity (from 10-12 to 10-18 mol), and range of accessible chemical and biological molecular families, it is very safe to predict that the impact of mass spectrometry will continue to increase exponentially well into the next millenium. Acknowledgment. The support of the National Science Foundation (M.T.B., A.G.M., F.W.M.), the Air Force Office of Scientific Research (M.T.B.), and the National Institutes of Health (A.G.M., F.W.M.) is gratefully acknowledged. References and Notes (1) Kroto, H. W.; Heath, J. R.; O’Brien, S. C.; Curl, R. F.; Smalley, R. E. Nature (London) 1985, 318, 162.

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