Ionization Mass Spectrometry

Applications of Laser Desorption and Electrospray Ionization Mass Spectrometry at the Transition between Clusters and Colloids. Paul J. Dyson, Brian F...
0 downloads 0 Views 117KB Size
Research: Science & Education edited by

topics in chemical instrumentation

Howard Strobel Duke University Durham, NC 27708-0354

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Instrumentation and Applications David C. Muddiman* Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284-2006 Ray Bakhtiar Department of Chemistry and Chemical Engineering, Stevens Institute of Technology, Castle Point on Hudson, Hoboken, NJ 07030 Steven A. Hofstadler and Richard D. Smith Macromolecular Structure and Dynamics Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352 Applications of mass spectrometry as an analytical method have grown in geometric progression since J. J. Thomson’s observation in the early 1900s that gaseous ions and neutrals (gaseous species without charge) could be produced by ion bombardment of samples and the ions separated according to their m/z ratio (1). Although early measurements provided invaluable information about stable isotopes and radionuclides, they were limited to compounds of low molecular weight that could readily be volatilized. More recently, volatilization–ionization methods have been developed that promote large, nonvolatile species into the gas phase as intact ions for mass spectrometric analysis. Independently, Hillenkamp and co-workers (2) and Tanaka and co-workers (3) introduced matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), which has significantly contributed to the field of biological mass spectrometry. This article introduces educators and researchers to the theory, principles, instrumentation, and some applications of MALDI-MS. Reflectron time-of-flight (TOF) mass analyzers are described in detail, since TOF is the most common mass analyzer for ions produced by MALDI. Several excellent reviews give a more detailed account of MALDI-MS (4–7). Mass Spectrometry The field of mass spectrometry has progressed significantly since Thomson’s development of the first mass spectrometer nearly a century ago. The remarkable improvement in instrumentation is largely attributable to technological advances and a more fundamental understanding of gas-phase ion behavior. However, in principle, all mass spectrometers have a common bond that has not changed over time: they measure the mass-to-charge (m/z) ratio of gaseous ions from a sample using electric or magnetic fields, where m is the ion mass in atomic mass units of daltons (Da) and z is the number of elemental charge units. A wealth of information (e.g., structure, purity of a chemical species, and composition) can be obtained solely from an accurate mass determination. Over the past two decades several new ionization techniques have been introduced, making possible the production of intact high-molecular-weight gas-phase ions of ther*Corresponding author.

1288

mally labile or nonvolatile compounds without derivatization: fast atom bombardment (FAB) (8), static secondaryion MS (SIMS) (9), plasma-desorption MS (PDMS) (10, 11), and laser desorption ionization (LDI) (12, 13). While these methods expanded the molecular weight range amenable to mass spectrometry, analysis of large biomolecules (> 5 kDa) was far from routine. Mass spectrometry using lasers for ion generation dates back to the 1960s (12). It was used initially to obtain elemental information and later for analysis of small biomolecules (13). However, an upper mass limit of 1000 Da was observed for biological molecules owing to excess transfer of laser energy to the analyte molecule (14). A milestone occurred in 1987 when MALDI was introduced by Hillenkamp and Karas, who used an organic matrix (2), and Tanaka and co-workers, who used an inorganic matrix (3). Because organic matrices have found wider applicability, they will be discussed in detail in this article. Since 1987, MALDI has been demonstrated to have several advantages, such as spectral simplicity owing to the production of singly charged ions larger than 500,000 Da with minimal observed fragmentation (15, 16). MALDI-MS has been used to investigate a wide range of compounds and biological systems, including nucleic acids (17–19), peptides and proteins (20–23), synthetic polymers (24–26), and drugs and metabolite systems (27, 28). The inherent “soft” ionization of MALDI allows promotion of these large thermally labile biological molecules into the gas phase without extensive fragmentation. The discussion of the MALDI experiment will include sample preparation and mass analysis of ions produced by MALDI. The often complimentary ionization technique, electrospray ionization (ESI), has been described elsewhere at a fundamental level (29). MALDI and ESI, which can be considered the foundation of the biological mass spectrometry era (now nearly a decade old), will be compared and contrasted below.

Feature Editor’s Note: This MALDI-MS paper is the fourth in a series of articles on mass spectrometry of very high molecular weight compounds. Earlier papers appeared in 1996 (J. Chem. Educ. 1996, 73, A82, A118, and A162) and dealt with electrospray ionization mass spectrometry. HS

Journal of Chemical Education • Vol. 74 No. 11 November 1997

Research: Science & Education The Matrix-Assisted Laser Desorption/Ionization Technique The MALDI process differs from direct laser desorption in that it directs a laser at an analyte imbedded in an excess of a specific matrix material. Figure 1 illustrates the overall process. From this perspective, MALDI is similar to fast atom bombardment (FAB), the latter utilizing liquid matrices to promote soft ionization. However, MALDI is unique in providing much softer ionization than FAB, which allows analysis of large nonvolatile molecules with minimal fragmentation. The matrix is the key component in MALDI-MS. Properties essential to a MALDI matrix are (i) high molar absorptivity at the laser wavelength employed; (ii) matrix solubility in the same solvent as the analyte material; (iii) appropriate physical properties such as lattice structure and heat of sublimation; (iv) ability to promote ionization; and (v) good vacuum stability. In practice, the matrix should provide maximum analyte ion intensity and signal reproducibility, and should minimize fragmentation and adduct formation with the biomolecule. It must also retain its properties in the presence of contamination. Although the above properties of a matrix are generally agreed upon, identification of new matrices that work well in MALDI has been slow even though most of the criteria are met. Because of the limited number of matrices available for use in MALDI, other parameters have been investigated, such as use of different substrates, development of multicomponent matrices and co-matrices, and different procedures for preparing the matrix/analyte mixture (e.g., how the two materials are mixed). Ionization of the biomolecule is thought to occur in the condensed phase or just above the surface in the volume of solid that was ablated by the laser pulse. It has been suggested that the matrix provides a source of gas-phase pro-

Figure 1. Analyte molecules are entrained in the crystal lattice of the matrix with some impurities remaining (e.g., Na +, K +). Upon irradiation of the solid matrix/analyte with a laser beam, matrix and analyte are ablated from the surface and extracted into a mass analyzer—in this case, a TOF mass spectrometer. The soft ionization is a result of the large excess of matrix (relative to analyte) having a high molar absorptivity coefficient and therefore absorbing the energy imparted by the laser.

tons for ionization (proton transfer reaction) in the positiveion mode. However, in some cases—even in the presence of the acidic matrix, oligosaccharides, and some other biopolymers—cationization with a metal (e.g., Na+, K +) is the preferred pathway for ionization (28, 30, 31). Clearly, the mechanism of ionization is still not completely understood.

Sample Preparation There are several methods of sample preparation, including the dried-droplet (2), fast evaporation (32), and smashed crystals (33) methods—all with numerous variations. Since the dried-droplet and fast-evaporation methods are the most commonly used (in part, owing to their simplicity), they will be described here. The matrix and analyte are dissolved in the appropriate mixture of organic solvent/water and either applied sequentially to a solid substrate or mixed together in a vial and deposited on the substrate (typically, polished stainless steel or nitrocellulose), where solvent evaporation occurs under ambient conditions or accelerated drying using compressed air or N2 . The molar ratio of analyte:matrix is usually 1:103 –105 ; clearly, there is a large molar excess of matrix. The matrix crystallizes upon drying and concurrently individual analyte molecules are isolated in it. While analyte molecules are not excluded, presumably owing to their much larger size (and thus lower diffusion constants), buffers can be partially “removed” during crystallization. Therefore, in theory, MALDI should be able to handle more complex sample matrices (e.g., alkali salts, buffers) such as those commonly used by biochemists. The major disadvantage of the dried droplet method is that it usually creates heterogeneous matrix crystals. This is important because the matrix crystal structure indicates the spectral quality. Some correlation between the point of laser/crystal interaction and spectral quality has been reported (34). It was suggested that one cause of the poor shotto-shot reproducibility sometimes observed is the presence of different sizes and shapes of crystals in the same sample. A wide range of analyte/matrix molar ratio distributions can be present within a single sample preparation (34). A sample preparation method termed “fast matrix evaporation” applies the matrix layer to the surface in a highly volatile solvent (acetone) to create a thin film of matrix (32). The key to this technique is creation of homogeneous crystals to which the analyte is deposited on top of a polycrystalline film. This method provides higher resolution and sensitivity than the dried-droplet method, while improving signal reproducibility. This has been attributed to the homogeneous crystal structure. Although most of the attention to sample preparation has focused on new matrix preparation techniques, the substrate upon which the matrix/analyte solution is deposited has also been investigated. Membrane supports including nylon (35), poly-(vinylidine difluoride) (36), nitrocellulose (37), and polyethylene (38) have been investigated. Possibly the most encouraging results relating to MALDI analysis of DNA are those obtained using nitrocellulose (39). An obvious question at this point is, how does one select the matrix, matrix preparation method, and substrate best suited for a particular analysis? The exact sample preparation method for MALDI analyses is highly sample specific. Thus, for a given biological system, an Edisonian approach (trial and error) is demanded. Determining the optimum sample preparation method can be laborious (an apparent disadvantage of MALDI), but once a suitable protocol is developed for the problem at hand, one can generally proceed using the established technique. After the crystallization step, the sample is inserted into the vacuum system of the spectrometer and pumped down to ca. 10 {8 torr. At this point, the laser is fired at the

Vol. 74 No. 11 November 1997 • Journal of Chemical Education

1289

Research: Science & Education surface, a volume of material is ablated (matrix and analyte), and positive or negative ions are mass analyzed. The most common lasers are N 2 gas and neodymium–yttrium garnet (Nd-YAG), whose fundamental frequencies are 337 nm and 1.064 mm, respectively. The YAG emission is usually frequency quadrupled and then emits at 266 nm. The laser power is usually normalized with respect to the irradiated area (ca. 50 µm); power densities are generally in the range of 5.0 × 107 W/cm 2. Multiple laser pulses (typically no more than 200) are usually averaged to improve the signalto-noise ratio.

Time-of-Flight Mass Analysis of MALDI-Produced Ions A time-of-flight (TOF) mass analyzer is commonly used for MALDI-MS because the pulses of ions generated in this way provide the sampling mode required by TOF analyzers. Advantages of TOF mass analyzers over other MS platforms include simplicity, high transmission, theoretically unlimited mass range, detection of all species simultaneously (multiplex advantage), high speed of analysis, and direct compatibility with pulsed ionization sources. Interestingly, although TOF analyzers are most suitable for pulsed ionization at solid surfaces, they are now being used for several continuous ionization sources such as capillary electrophoresis electrospray mass spectrometry (40). In MALDI, the ions are created by a laser pulse that strikes the matrix-covered surface. To measure the time-offlight of a particular ion, the TOF “clock” needs to be set like a stopwatch (i.e., t = 0 needs to be defined). This can be accomplished in several ways: when the laser is fired, when a photodiode placed near the sample is activated by the laser pulse, or when the mass spectrometer extraction voltage is turned on. Instrument configurations differ; a desirable method is one that reduces jitter, since multiple pulses are averaged together. If the jitter is too large, mass resolution will be poor. The ions formed from the laser pulse are extracted into the TOF analyzer by an electrical field (Uex). The kinetic energy (KE) given to an ion by the electric field is the product of the charge of the ion and the electrical field strength: KE = z e Uex

are known: t = ∆x (m / 2 z e Uex) 1/2

(5)

For example, the flight time of a singly charged ion (z = 1) of horse-heart cytochrome c (MW = 12,361), assuming the spectrometer has a 1.2-m flight path and the ions are being extracted at 5 kV, will arrive in 136 µs. In more convenient terms, eq 4 can be written so that m is the molecular mass of the ion in daltons; and since 1 Da is equal to 1.660 × 10{27 kg and e = 1.60 × 10 {19 C, the equation can be written m / z = (9.638 × 107 ) 2Uex (∆t2/∆x2)

(6)

The production of primarily singly charged ions (z = 1) in MALDI clearly demands high m/z mass analyzers. As previously mentioned, TOF has a theoretically unlimited mass range (e.g., if a Boeing 747 enters into one end of an empty tube a Boeing 747 will come out the other end— assuming no collisions!) This places stringent requirements on the detection system because they are velocity-dependent (velocity-sensitive) detectors. This can be seen rearranging eq 3: v = (2z e Uex/m) 1/2

(7)

Thus the velocity decreases inversely with the square root of the mass, and therefore the sensitivity is reduced for high molecular weight species. Because of the large kinetic energy difference of the ions produced at the surface, linear TOF analyzers do not afford high mass resolution because the initial kinetic energy distribution (which increases with mass) is not being compensated for. The reflectron TOF configuration was introduced in 1966 (41) and several variations have subsequently appeared. The reflectron-TOF (reTOF) consists of two linear field-free regions and an ion mirror (depicted in Fig. 2), which compensates for different, nonzero initial kinetic energies of ions of the same m/z. Figure 2 depicts what occurs if two ions (1 and 2) of the same mass but different initial kinetic energy (E1 > E2)

(1)

where z is the charge of the molecular ion, e is the charge of an electron in coulombs, and Uex is the electric field strength in volts. In the ideal case, all ions (regardless of mass) enter the field-free region with identical kinetic energy. From Newtonian mechanics, we know the following relationship: KE = (1/ 2) mv2 = (1/ 2) m (dx/dt)2

(2)

Therefore the right side of eq 1 is equal to the right side of eq 2: z e Uex = (1/ 2) m (dx/dt)2

(3)

Since the ions are extracted in a very short “source region” relative to the time they spend in flight, the velocity is a constant in these equations; thus, we can replace the instantaneous velocity with the total flight time (∆t) and the total field-free flight path (∆x). By rearranging eq 3, m/z for a given ion can be determined: m /z = 2 e Uex ∆t 2/∆x2

(4)

where m is the mass in kilograms, ∆x is the flight path length of the ion in meters, and ∆t is the flight time in seconds. The total flight time is tarrival – t0; thus, ∆t becomes t. Equation 4 can also be written to determine the arrival time of a given molecular ion if the experimental conditions

1290

Figure 2. Schematic of a reflectron TOF mass analyzer. Two ions of the same mass but different initial kinetic energies, E1 and E2 , are produced and extracted into the first field-free region, L1. If the TOF is operated in the linear mode, a mass spectrum of poor resolution results (top right). However, if the linear detector is bypassed by utilizing the ion mirror, the resolution is markedly enhanced (lower left).

Journal of Chemical Education • Vol. 74 No. 11 November 1997

Research: Science & Education are desorbed from a surface and mass analyzed by a reTOF. Ions extracted from the surface (Uex) traverse the first fieldfree region (L 1). Thus, the kinetic energy of ion 1 is (Uex + E1 ) and of ion 2 is (Uex + E2). The time required for each ion to arrive at the entrance to the ion mirror can be determined: t = L1 / v ≅ 1 / KE(1/2)

(8)

where t is the time, L1 is the length of the first linear region, v is the velocity of the ion, and KE is the total kinetic energy of the ion. If E 1 has a greater kinetic energy than E2 , E 1 will traverse the field-free region first. This is illustrated in Figure 2, with the brackets indicating the pair of ions that originated at the surface from the desorption laser pulse. In the reflecting region (R), the ion with the greater kinetic energy (E 1) will penetrate further into the ion mirror, as shown in Figure 2. The distance the ion travels into the ion mirror, which is proportional to flight time, depends on the ion’s energy. Thus, the ion with the initial kinetic energy E1 will traverse further into the field than the ion of energy E2 and will therefore spend a longer time in region R. When the ions leave the reflecting field they enter into a second field-free linear region (L 2) and will ultimately strike the detector. However, note that the ion with energy E2 is in front of ion E1 at the exit of the ion mirror even though E 1 is still greater than E 2. Time focusing is achieved by changing R (the length of the reflecting region) by altering the voltage of the ion mirror, since it is not experimentally feasible to change L 1 and L 2. As the ions travel through the second field-free region (L2 ), ions E1 and E 2 become “time focused” at the plane of the detector because ion E1 “catches up” to ion E 2 owing to its greater kinetic energy. This “time focusing” phenomenon results in significantly improved mass resolution, as depicted in Figure 2. Recently, the implementation of delayed ion extraction, an important innovation originally proposed by Wiley and McLaren (42), has greatly improved mass resolution (43, 44). Delayed extraction allows the ions to disperse in the source region (acceleration region just above the surface) owing to their initial velocity, while the density of neutrals is decreased by pumping them away. The number of ion– molecular collisions before extraction of the ions into the field-free drift region is therefore reduced, which decreases the width of the translational energy distribution. Simulta-

Figure 3. MALDI-TOF mass spectrum (reflectron mode) of the Na + cationized oligomer distribution of poly(butylene adipate) (PBA) using a trans-3-indoleacrylic acid matrix. The inset shows the structure of the polymer, indicating the repeat unit and terminal group.

neously, ions having higher initial kinetic energy move further away from the extraction field in the source region and thus are given less “kick”; those of lower kinetic energy experience a higher extraction field. This approach has proven so successful that limitations in mass resolution are now focused on detection systems (the time resolution). Applications of MALDI It is evident from the vast amount of literature that MALDI is playing an ever-increasing role in biological chemistry. No single article could adequately “review” the most important applications of MALDI and would certainly prejudice the reader. Thus, we will show only a single application of MALDI that demonstrates arguably one of its most important features: the production of primarily singly charged ions, allowing the analysis of extremely heterogeneous mixtures. We will conclude this section with a brief comparison of MALDI with ESI. The following application is related to synthetic polymer analysis using MALDI-TOF. Figure 3 is a spectrum of a synthetic polymer, polybutylene adipate; the oligomer distribution (a condensation synthesis results in a high polydispersity) is easily resolved and an average molecular mass of 4525 Da is calculated. Since the ions are all singly charged, the distance between oligomers (∆) is equal to the mass of the repeating unit (200 Da). It is clear that MALDITOF offers a unique and facile approach to molecular weight characterization of complex samples—in this case, the average molecular weight of the polymer, which is known to correlate to the properties of the polymer. In addition, the mass of terminal group of the polymer can be readily deduced: mass of terminal group = measured MW of oligomer – (n)(∆)

where n is the number of repeat units in the oligomer and ∆ is the mass of the repeat unit. Another application demanding spectral simplicity (or high resolution) would be the analysis of DNA sequencing ladders. An obvious concluding question is, how can I decide whether to use MALDI or ESI to address a particular scientific problem? While a problem might fall into a general category such as protein analysis, each individual problem has such specificity that no overview could possibly detail all the reasons to use one or the other. For example, there are many types of proteins (transmembrane proteins, which are hydrophobic, glycoproteins, etc.) and many types of information can be sought (e.g., primary sequence, higher-order structure, site of glycosylation); therefore, each problem dictates a different approach. For example, a 45-kDa protein may not carry adequate charge during ESI and will not fall within the m/z range of a linear quadrupole. In this case, MALDI-TOF (with an unlimited m/z range) might be the best analytical approach. An approach not only entails the type of ionization technique but also the mass analyzer, the MALDI matrix or the composition of the solution to be infused (ESI). Thus, knowledge of the “bulk” properties and capabilities of each method should help in establishing the appropriate experimental design. The general characteristics of MALDI and ESI are summarized in Table 1. Note that roughly the same percentage of work is conducted on peptides, proteins, and nucleic acids by MALDI and ESI; however, ESI is more commonly employed to investigate small molecules. It is evident from Table 1 that MALDI and ESI are truly complementary in nature, almost a mirror image being projected between the disadvantages of one technique and the advantages of the other, while both provide “unlimited” mass range and high analyte sensitivity.

Vol. 74 No. 11 November 1997 • Journal of Chemical Education

1291

Research: Science & Education Table 1. Comparison of MALDI and ESI Mass Spectrometry Feature

MALDI-MS

Ionization

Pulsed

Continuous

Common mass analyzers

TOF, FTICR, QITa

Quad, QIT, FTICR, TOF, Sectora

Mass range

>1,000,000 Da

>1,000,000 Da

Detection limits

femtomole–attomoleb

femtomole–zeptomoleb

Sensitivity to

contaminationc

ESI-MS

reasonably tolerant

reduces sensitivity dramatically

Spectral simplicity

production of primarily singly charged ions allows analysis of complex mixtures at relatively low mass resolution

analysis of mixtures is difficult because each compound gives rise to a charge-state distribution

Coupling with on-line separations

difficult in most cases

facile

a TOF: time-of flight; FTICR: Fourier transform ion cyclotron resonance; QIT: quadrupole ion trap; QUAD: linear quadrupole; Sector: magnetic sector. b Best detection limit reported to date but not routinely obtainable. cContamination: e.g., buffers, salts, cellular components.

Conclusions Since the pioneering efforts of Biemann (45), the biological mass spectrometry era has flourished and had a remarkable impact in biomedicine and biochemistry. This rapid growth can be directly correlated to the introduction of soft ionization techniques—most notably, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Both MALDI and ESI have been coupled with a variety of mass analyzers and have solved or provided a deeper insight into diverse biological problems. The limits of both techniques have yet to be defined, and they are certain to play an ever-increasing role in multidisciplinary research. Acknowledgments D.C.M. gratefully acknowledges Virginia Commonwealth University for financial support and Kimberly E. Muddiman for her help in preparing the manuscript. The authors acknowledge John Williams (University of Pittsburgh) for providing the MALDI-TOF spectrum of the polymer and Harold Udseth (Pacific Northwest National Laboratory) for helpful discussions. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy, through contract DE-AC0676RLO 1830. Literature Cited 1. Thomson, J. J. Philos. Mag. 1910, 20, 752. 2. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. 3. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. 4. Karas, M.; Bahr, U.; Giessmann, U. Mass Spectrom. Rev. 1991, 10, 335–357. 5. Hillenkamp, F. Adv. Mass Spectrom 1995, 13, 95–114. 6. Mann, M.; Talbo, G. Curr. Opin. Biotechnol. 1996, 7, 11–19. 7. Muddiman, D. C.; Gusev, A. I.; Hercules, D. M. Mass Spectrom. Rev. 1995, 14, 383–429. 8. Barber, M.; Green, B. N. Rapid Commun. Mass Spectrom. 1987, 1, 80–83. 9. Benninghoven, A. Angew. Chem. Int. Ed. Engl. 1994, 33, 1023–1043. 10. Macfarlane, R. D.; Torgerson, D. F. Science 1976, 191, 920. 11. Sundqvist, B.; Macfarlane, R. D. Mass Spectrom. Rev. 1985, 4, 421–460. 12. Vastola, F. J.; Mumma, O.; Pirone, A. J. Org. Mass Spectrom. 1970, 3, 101. 13. Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C.; Ten Noever deBrauw, M. C. Anal. Chem. 1978, 50, 985. 14. Cotter, R. J. Anal. Chim. Acta. 1987, 195, 45–59. 15. Nelson, R. W.; Dogruel, D.; Williams, P. Rapid Commun. Mass Spectrom. 1995, 9, 625.

1292

16. Imrie, D. C.; Pentney, J. M.; Cottrell, J. S. Rapid Commun. Mass Spectrom. 1995, 9, 1293–1296. 17. Hillenkamp, F. Biomed. Health Res. 1995, 8, 198–205. 18. Bai, J.; Lin, Y. H.; Liang, X. L.; Zhu, Y. D.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 1172–1176. 19. Liu, Y.-H.; Bai, J.; Zhu, Y.; Liang, X.; Siemieniak, D.; Venta, P. J.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 735– 743. 20. Bahr, U.; Karas, M.; Hillenkamp, F. Fresenius J. Anal. Chem. 1994, 348, 783–791. 21. Blais, J. C.; Nagnanlemeillour, P.; Bolbach, G.; Tabet, J. C. Rapid Commun. Mass Spectrom. 1996, 10, 1–4. 22. Brown, R. S.; Carr, B. L.; Lennon, J. J. J. Am. Soc. Mass Spectrom. 1996, 7, 225–232. 23. Strupat, K.; Karas, M.; Hillenkamp, F.; Eckerskorn, C.; Lottspeich, F. Anal. Chem. 1994, 66, 464–470. 24. Danis, P. O.; Karr, D. E.; Mayer, F.; Holle, A.; Watson, C. H. Org. Mass Spectrom. 1992, 27, 843–846. 25. Danis, P. O.; Karr, D. E.; Simonsick, W. J.; Wu, D. T. Macromolecules 1995, 28, 1229–1232. 26. Danis, P. O.; Karr, D. E.; Xiong, Y. S.; Owens, K. G. Rapid Commun. Mass Spectrom. 1996, 10, 862–868. 27. Muddiman, D. C.; Gusev, A. I.; Proctor, A.; Hercules, D. M.; Venkataramanan, R.; Diven, W. Anal. Chem. 1994, 66, 2362–2368. 28. Muddiman, D. C.; Gusev, A. I.; Stoppeklangner, K.; Proctor, A.; Hercules, D. M.; Tata, P.; Venkataramanan, R.; Diven, W. J. Mass Spectrom. 1995, 30, 1469–1479. 29. Hofstadler, S. A.; Bakhtiar, R.; Smith, R. D. J. Chem. Educ. 1996, 73, A82. 30. Harvey, D. J. Rapid Commun. Mass Spectrom. 1993, 7, 614–619. 31. Harvey, D. J. J. Chromatogr. 1996, 720, 429–446. 32. Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281–3287. 33. Xiang, F.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 199–204. 34. Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Anal. Chem. 1995, 67, 1034–1041. 35. Zaluzec, E. J.; Gage, D. A.; Allison, J.; Watson, J. T. J. Am. Soc. Mass Spectrom. 1994, 5, 230–237. 36. Vestling, M. M.; Fenselau, C. Mass Spectrom. Rev. 1995, 14, 169– 178. 37. Preston, L. M.; Murray, K. K.; Russell, D. H. Biol. Mass Spectrom. 1993, 22, 544–550. 38. Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843– 848. 39. Liu, Y. H.; Bai, J.; Liang, X. O.; Lubman, D. M.; Venta, P. J. Anal. Chem. 1995, 67, 3482–3490. 40. Muddiman, D. C.; Rockwood, A. L.; Gao, Q.; Severs, J. C.; Udseth, H. R.; Smith, R. D.; Proctor, A. Anal. Chem. 1995, 67, 4371–4375. 41. Mamyrin, B. A.; Karateev, V. I.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys. JETP 1973, 37, 45. 42. Wiley, M. C.; McLaren, I. H. Rev. Sci. Instrum. 1955, 26, 1150. 43. Anderegg, R. J.; Wagner, D. S.; Stevenson, C. L.; Borchardt, R. T. J. Am. Soc. Mass Spectrom. 1994, 5, 425–433. 44. Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044–1050. 45. Biemann, K. Annu. Rev. Biochem. 1992, 61, 977–1010.

Journal of Chemical Education • Vol. 74 No. 11 November 1997