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[71 J.E. Campana (Editor), in Time-of-Flight Mass Spectrometry; Special issue of Anal. Instrum., Marcel Dekker, New York, NY, 1987, Vol. 16, No. 1. [81 D. Price and G.J. Mimes, Int. J. Mass Spectrom. Zen Processes, 99 ( 1990) l-39. (Editor), Lasers and Mass L91 D.M. Lubman Spectrometry (Oxford Series on Optical Sciences), Oxford University Press, New York,

NY, 1990. [lOI D.A. Laude, Jr. and J.D. Hogan, Technisches Messen, 57(4) (1990) 1.55-159. [Ill C.E. Brown, M.J.C. Smith, Spectrosc. World, 2( 1) (1990) 24-30. [ 121 A.G. Marshall and L. Schweikhard, Znt. J. Mass Spectrom. Ion Processes, 118/ 119 ( 1992) 37-70. [I31 C. Koster, M.S. Kahr, J.A. Castor0 and C.L. Wilkins, Mass Spectrom. Rev., 11 (1992) 495-

512.

[ 141 P. Kebarle and L. Tang, Anal. Chem., 65 (1993)

972A-986A. [ 151 S.C. Beu, M.W. Senko, J.P. Quinn and F.W. McLafferty, J. Am. Sot. Mass Spectrom., 4 ( 1993) 190-192. [ 161

L.-S. Sheng, S.L. Shew, B.E. Winger and J.E. Campana, in T. Provder, M. Urban and H.G. Barth (Editors), Hyphenated Techniques in Polymer Characterization (American Chemical Society Symposium Series), American Chemical Society, Washington, DC, 1994.

Joseph E. Campana, Long-Sheng Sheng, Sanford L. Shew and Brian E. Winger are at Extrel FTMS, Waters, 6416 Schroeder Road, Madison, WI 5371 l-2424, USA.

Electrospray ionization with Fourier transform mass spectrometry Evan R. Williams Berkeley,

CA, USA

Electrospray ionization (ESI) has become widely used to solve problems in many diverseareas of chemistry and biochemistry. In combination with Fourier transform mass spectrometry (FTMS), even more chemical information can be obtained from trace quantities of complex samples, including those present in mixtures. This article reviews some of the exciting capabilities, instrumentation, and selected applications of ESIFTMS.

1. Introduction Mass spectrometry (MS) is becoming an increasingly powerful method for detection and characterization of large biomolecules, especially those present at trace levels and in complex mixtures. Two ionization techniques, matrixassisted laser desorption/ionization and electrospray ionization, have greatly extended the mass 8 1994 Elsevier Science B.V. All rights reserved

range of MS [ 11. These methods produce intact, gas-phase molecular ions from molecules in the hundreds of thousands, even millions of molecular mass. From these ions, molecular masses can be measured with unprecedented accuracy (e.g., < 0.01%) For mass spectrometers with an upper m/z limit, electrospray ionization has the advantage that multiply charged ions are produced [ 21. This reduces the m/z ratio of molecular ions to cu. 500-2500, a range in which the performance of many instruments, such as the Fourier transform (FT) mass spectrometer, excels [ 3,4]. For characterization of these multiply charged ions, FTMS has many exciting features, including multichannel, non-destructive detection (recording a complete spectrum of all ions simultaneously), ultra-high mass resolution ( > lo6 at m/z 1000) and extensive tandem MS (even MS”) capabilities. Instrumentation and selected applications of ES1 with FTMS are presented here; this review is not comprehensive, but rather highlights some of the exciting recent advances in this field. 2. Instrumentation In order to take full advantage of the high performance capabilities of FTMS, namely ultrahigh 01659936/94/$07.00

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In a novel ‘internal’ electrospray source, developed by Hofstadler and Laude [ 13,141, the atmospheric pressure ES1 source is placed inside the bore of the magnet approximately ten centimeters from the cell. Although pumping conductance in this configuration is reduced by the confines of the magnet, this approach has the advantage that the electrospray process takes place in the high magnetic field. This helps confine the ions in the radial direction, enabling more ions to pass through the interface into the ion cell.

resolution and long ion storage times, low ion cell pressure (preferably < 1O-8 torr) must be maintained. This presents a key difficulty in combining ESI, primarily an atmospheric pressure technique, with FTMS. A number of innovative solutions to this problem have been developed [ 5-141. External ion source instruments, in which ions are formed outside the confines of a (superconducting) magnet, have been developed by a number of groups [ 5-121. This approach provides open pumping conductance for differentially reducing pressure between the ion source and FTMS cell. With these external source instruments, ions must be guided past the fringing fields of the magnet, a task complicated by the infamous ‘magnetic mirror’ effect [ 81. McLafferty, Hunt and co-workers [ 91 first demonstrated the combination of ES1 with FTMS, using an external source and quadrupole ion guides to inject ions into the cell. Subsequent quadruple introduction systems [ 10,l 1 ] have proven to be a tremendously successful approach for combining ES1 with FTMS. We have developed, in collaboration with Extrel FTMS Millipore Corp., anew external electrospray ion source FTMS instrument that employs electrostatic lenses that accelerate ions to cu. 2.2 kV to guide them past the fringing fields of the magnet and decelerate them back to ion source potential prior to injection into the cell (Fig. 1) . This system has the advantage that little radial (cyclotron) energy is imparted to the ions upon injection into the cell. A version of this system (UltraSource FTMS, Extrel FTMS Millipore Corp., Madison, WI, USA), as well as the BioAPEX (Bruker Instruments, Billerica, MA, USA), an external ion source instrument that also uses electrostatic lenses, are commercially available [ 121.

3. Performance 3.1. Sensitivity Truly outstanding performance in terms of resolution and sensitivity has been obtained with each of these systems. Acquisition of complete mass spectra of biomolecules from low femtomole and even attomole quantities of material consumed has been demonstrated [ 1O-141. It should be noted that these values correspond to the amount of sample introduced into the mass spectrometer during an ion accumulation event. This can be quite short, often just a few milliseconds. Thus, these values represent the potential of this instrumentation for high sensitivity rather than the absolute amount of material required for ‘real world’ acquisition of mass spectra. For the full potential in sensitivity to be reached, improvements in ion trapping and matching the ion collection duty cycle with that for sample introduction is required. An important advantages of the FTMS detection over conventional ion detection methods, such as electron multipliers, is that FTMS detection is nonExternal Source Electrospray Ionization

2.7 Tesla Superconducting Magnet ion guides

to syringe pump or CE

diffusion ion pump

pumps

’ + diffusion

Fig. 1. Schematic of our external ion source ESI-FTMS atmospheric pressure source into the FTMS ion cell.

pumps

pump instrument;

electrostatic

lenses

guide

ions from the

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destructive; after detection, ions can be collisionally relaxed back to the center of the cell where they are available for subsequent manipulation, e.g. dissociation or reaction, and remeasurement [ 1.51. Laude and co-workers [ 161 demonstrated that a 16-fold improvement in signal-to-noise for bovine albumin dimer ions (M, 133 kDa) could be obtained by remeasuring the same ions 250 times. This signal gain corresponds to a 100% remeasurement efficiency. Relaxation of these large ions to the center of their orbits was found to occur within a few hundred milliseconds after excitation. An FTMS signal is proportional to the charge of an ion. With ESI, charge increases with increasing mass. Thus, large multiply charged ions are more readily detected than smaller, less highly charged ions! Thus, in principle, the mass range of ESIFTMS is virtually unlimited (vi& infra).

3.2. Resolution

Another key feature of FTMS detection is that it is nearly independent of ion energy; more energetic ions have larger cyclotron orbits, but the same cyclotron frequency. This makes possible high resolution detection of ions, even after excitation or dissociation. Resolution of up to 3. lo6 [ 10-l 21 has been demonstrated for proteins below 30 kDa. With this high resolution, ion isotopic envelopes are readily resolved making possible the accurate measurement of isotope distributions from these large ions. This is particularly useful for separating mixtures of molecules differing in mass by a few hundredths of a percent. Mass measuring accuracies of a few parts per million (ppm) are routinely obtained; with careful attention to calibration, an impressive mass measuring accuracy of 0.15 ppm has been demonstrated by McLafferty and coworkers [ 171 for myoglobin (M, 16 946.98 Da for ‘Q. Extending these measurements to molecules larger than 100 kDa is complicated by the limited dynamic range of FTMS; more than lo5 or so charges present in the cell produce sufficient Coulomb forces between ions to significantly reduce resolution. For 200 kDa ions with 200 charges each, this corresponds to approximately 500 ions. This number is insufficient to obtain accurate isotopic abundances in a single measurement. In addition, sample microheterogeneity can further exacerbate such measurements.

4. Selected applications 4.1. Separations with ESI-FTMS A number of methods for separating biomolecule mixtures have been developed. For combining these techniques with MS detection, microbore HPLC and capillary electrophoresis (CE) have the advantage that the low flow-rates used with these methods are well suited to the electrospray process. This is of particular concern in cases where sample quantity is limited, e.g., natural products characterization. The successful combination of CE with ESI-FTMS has been demonstrated by Smith and co-workers [ 18,191 with a six-component synthetic mixture containing somatostatin, lactalbumin, ubiquitin, carbonic anhydrase, myoglobin and lysozyme. From an injection corresponding to approximately 6 fmol of each component, complete mass spectra with excellent signal and resolution were obtained for each of the individual components. With the anticipated improvements in trapping efficiency and sample utilization, complete mass spectra from low and even sub-attomole quantities of biomolecules present in complex mixtures should be readily obtainable in the near future. 4.2. Protein and DNA sequencing Tandem mass spectrometry, in which an ion is mass selected, dissociated, and product ions mass analyzed, has been extensively used for sequencing small peptides ( I 3000 Da) of unknown structure. The large multiply charged ions produced by electrospray ionization can be dissociated readily, producing fragment ions indicative of ion structure. However, mass spectrometers measure m/z ratios; determining the mass of fragment ions of multiply charged precursors is complicated by the number of charges possible on a fragment ion. As first demonstrated by McLafferty and co-workers [ 201, the charge of an ion can be readily determined if the isotopic peaks, separated in mass by one dalton, are resolved. For example, an ion cluster with 7 isotope peaks per unit m/z, has 7 charges. Thus, high resolution tandem MS detection enables the charge, and hence the mass of each fragment, to be easily measured. In combination with accurate molecular mass measurements, tandem MS can be tremendously useful for confirming ion structure. By comparing the masses of fragment ions from a protein of

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known structure to those of a protein whose sequence is suspect, deviations, such as point mutations in a protein, can be located. In addition, tandem MS experiments can be done rapidly ( < 1 min), and lends itself to applications such as quality control of engineered biomolecules. The potential of tandem MS for obtaining sequence information on proteins of unknown structure is beginning to be explored. For the dissociation of carbonic anhydrase (M, 29 kDa, whose structure is already known), McLafferty and co-workers [ 2 I] obtained the masses of over 100 isotopic fragment clusters from their high resolution measurements. Approximately 80% of these products were formed by backbone cleavage near the amino acid proline. Additional stages of MS (MS’“) could be used to obtain sequence information from each of these fragment ions. In theory, the complete sequence of a protein of unknown structure could be obtained from such experiments. 4.3. Ion chemistry Ions can be stored for several minutes,

even hours in the FTMS cell. This ion storage capability is ideally suited for studying the chemistry of gasphase ions. An elegant example of the kind of information that can be obtained from these experiments has been reported by McLafferty and co-workers [22] who used gas-phase deuterium exchange experiments to differentiate different conformations of proteins in the gas-phase. For example, these researchers found three distinct gas-phase conformers of cytochrome c whose reactivity was consistent with known solution-phase conformations. Those conformers with higher numbers of reactive hydrogens were attributed to more open structures that have increased accessibility of exchange sites. In striking contrast, deuterium exchange experiments by Smith and co-workers [ 231 in the higher pressure interface region, indicate that ions with more native conformations exchange more hydrogens. The authors attributed this to the increased charge-charge repulsion, or Coulomb energy, in these more compact structures, resulting in increased reactivity. Our laboratory has recently demonstrated a method to quantitatively measure the Coulomb energy in multiply charged ions through measurement of their gas-phase basicity [ 241. From these measurements, fundamental properties of these molecules, such as the intrinsic dielectric polarizability, as well as distance between charges can be

trends in analytical chemisfry, vol. 13, no. 6, 1994

obtained. This latter value should provide a useful measure of the shape of folded gas-phase protein ion structure. Future work measuring gas-phase biomolecule reactivity and comparing these results with those in solution is an exciting new area to be explored.

5. Future developments The successful application of mass spectrometry for solving problems of biochemical and biomedical interest has led to an explosive growth in electrospray ionization over the last several years. The combination of ES1 with FTMS has many exciting advantages for extracting even more information from complex samples, and makes possible many new experiments. For example, following nondestructive FTMS detection, individual highly charged ions of large molecules could be reacted or dissociated and the resulting product ion or ions measured to obtain structural information. This has been proposed as a method to sequence an individual DNA molecule [ 191. In theory, ions in the hundred million molecular mass range could be analyzed with this technique. Characterization of the conformation and reactivity of gas-phase biomolecule ions could improve understanding of the role of solvent in protein structure and function, and provides complementary information to that obtained using more traditional techniques such as crystallography or NMR.

Acknowledgements Support of the National (CHE-92.58 178)) the Arnold Foundation (M 1652)) the ( 13605) and Extrel FTMS, gratefully acknowledged.

Science Foundation and Mabel Beckman Exxon Foundation Millipore Corp. is

References S.B.H. Kent, Science, 257 ( 1992) 1885-l 894; and references cited therein. [ 21 J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, Science, 246 ( 1989) 64-71; and references cited therein. [ 31 A.G. Marshall and P.B. Grosshans, Anal. Chem., 63 ( 1991) 215A-229A; and references cited therein. [ 1 ] B.T. Chait and

trends in analytical chemistry, vol. 13, no. 6, 1994

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[41 C. Koster, MS. Kahr, J.A. Castor0 and C.L. Wilkins, Mass Spectrom. Rev., 11 (1992) 4955 12; and references cited therein. [51 R.T. McIver, Jr., R.L. Hunter and W.D. Bowers, Int. J. Mass Spectrom. Ion Processes, 64 (1985) 67-77. [61 P. Kofel, M. Alleman, Hp. Kellerhals and K.P. Wanczek, lnt. J. Mass Spectrom. Ion Processes, 65 (1985) 97-103. [71 J.M. Alford, P.E. Williams, D.J. Trevor and R.E. Smalley, Int. J. Mass Spectrom. Ion Processes, 72 (1986) 33-51. [81 R.T. McIver, Jr., ht. J. Mass Spectrom. Zen Processes, 98 ( 1990) 35-50. [9 K.D. Henry, E.R. Williams, B.H. Wang, F.W. McLafferty, J. Shabanowitz and D.F. Hunt, Proc. Natl. Acad. Sci. USA, 86 (1989) 9075-9078. [lo SC. Beu, M.W. Senko, J.P. Quinn, F.M. Wampler, III and F.W. McLafferty, J. Am. Sot. Mass Spectrom., 4 (1993) 557-565. B.W. Winger, S.A. Hofstadler, J.E. Bruce, H.R. [111 Udseth and R.D. Smith, J. Am. Sot. Mass Spectrom., 4 ( 1993) 566-577. [I21 L. Voress, Anal. Chem., 66 ( 1994) 481486A. [I31 S.A. Hofstadler and D.A. Laude, Jr., Anal. Chem., 64 (1992) 569-572. [ 141 S.A. Hofstadler and D.A. Laude, Jr., J. Am. Sot. Mass Spectrom., 3 ( 1992) 615-623.

[ 151 E.R. Williams, K.D. Henry and F.W. McLafferty, J. Am. Chem. Sot., 1 I2 (1990) 6157-6162. [ 161 Z. Guan, S.A. Hofstadler and D.A. Laude, Jr., Anal. Chem., 65 (1993) 1588-1593. [ 171 S.C. Beu, M.W. Senko, J.P. Quinn and F.W. McLafferty, J. Am. Sot. Mass Spectrom., 4 (1993) 190-192. [I81 S.A. Hofstadler, J.H. Wahl, J.E. Bruce and R.D. Smith, J. Am. Chem. Sot., 115 ( 1993) 69836984. [I91 R.D. Smith, J.H. Wahl, D.R. Goodlett and S.A. Hofstadler, Anal. Chem., 65 ( 1993) 574584A. 1201 K.D. Henry and F.W. McLafferty, Org. Mass Spectrom., 25 ( 1990) 490-492. [211 M.W. Senko, S.C. Beu and F.W. McLafferty, Anal. Chem., 66 ( 1994) 415417. [221 D. Suckau, Y. Shi, S.C. Beu, M.W. Senko, J.P. Quinn, F.M. Wampler, III and F.W. McLafferty, Proc. Natl. Acad. Sci. USA, 90 ( 1993) 790-793. ~231 B.E. Winger, K.J. Light-Wahl, A.L. Rockwood and R.D. Smith, J. Am. Chem. Sot., 114 ( 1992) 5897-5898. v41 D.S. Gross and E.R. Williams, J. Am. Chem. Sot., submitted for publication. Evan R. Williams is an Assistant Professor of Chemistry in the Chemistry Department at the University of California, Berkeley, CA 94720- 1460, USA.

Laser methods in mass spectrometry Laser Ionization Mass Analysis, edited by A. Vertes, R. Gijbels and F. Adams, Wiley, Chichester, 1993, f 79.00 (xviii + 3 18 pages), ISBN O-471 -536-733

In recent years, the application of lasers in the field of mass spectrometry has led to a dramatic increase in the range of molecular masses that can be measured using this technique. This success has established laser methods in an area in which they had previously occupied only a peripheral role. Further advances are to be expected from current research into the practical and theoretical problems that arise from studies of how intense laser beams interact with organic and inorganic

compounds within a mass spectrometer. It is to those engaged in this research that this book is recommended. The book is divided into five chapters, the first of which is a brief introduction by the editors who also make other contributions later on. The remaining four chapters are lengthy and are the work of a total of thirteen authors. Generally, the quality of presentation is high and uniform in style. However, an anomaly is to be found in the title which refers only to laser ionization. Fortunately, the book is as much concerned with laser desorption. Chapter 2 gives a comprehensive survey of early work and describes ways in which lasers have been interfaced to magnetic sector instru-

ments, time-of-flight, quadrupole and, more recently, Fourier transform and ion trap mass analysers. Both prototype and commercially available instruments are described and the results obtained at different laser wavelengths and irradiances are discussed. Chapter 3 is subdivided into three sections each with its own list of references at the end. This chapter is concerned with work done at low and medium laser irradiance. Section A discusses desorption/ionization mechanisms and includes a description of work done on the matrix assisted laser desorption of proteins, DNA, carbohydrates and synthetic polymers. Section B describes work done on relatively small organic molecules including