Combinatorial chemistry methods

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1 I M A G I N G Imaging Mass Spectrometry and Combinatorial Chemistry Nicholas Winograd and obert M. Braun Penn State University University Park, PA Combinatorial chemistry methods use a series of reaction steps with multiple reagents for each step to create a collection of as many as several million compounds. The ability to create such chemical diversity is inspiring enthusiasm for novel drug discovery strategies in the pharmaceutical industry. For libraries of this size, the syntheses are usually carried out either on functionalized silicon wafers or on polystyrene spheres with diameters of 2 5 m. From a drug discovery point of view, the idea is to massively bioassay Speed is of the essence in screening diverse libraries of molecules for drug efficacy. Spatially resolved time-offlight mass spectrometry potentially offers chemical analysis at the rate of 1 compounds per second or more and is amenable to microarray formats. Liquid-metal ion gun Ga eflect etard Extraction optics Laser beam Sample stage Ion gun Stage voltage Laser Charge compensation Figure 1. Schematic diagram of a TOF-SIMS apparatus. 14 SPECTOSCOPY 16(9) SEPTEMBE 21

2 I M A G I N G the entire library. The active compounds are then identified as leads after release from the beads, and their structure may be identified by spectroscopic means. Mass spectrometry (MS) has become an essential element in the repertoire of tools available for the characterization of combinatorial libraries. The strategies range from traditional mass spectral measurements on bulk solutions for structural studies to identification of molecules delinked from a single polystyrene bead. Most types of MS, including matrixassisted laser desorption ionization (MALDI), electrospray, liquid chromatography MS (LC-MS), and Fourier transform infrared spectroscopy (1 5), have been brought to bear on the problem. The popularity of these methods stems in part from the chemical specificity, sensitivity, and speed of MS. Our laboratory has been exploring the use of imaging time-of-flight secondary ion MS (TOF-SIMS) as a means to further extend the applicability of these sorts of measurements (6). With this approach, molecules are desorbed from a solid surface by a pulsed energetic ion beam that is focused to a spot size of 1 m in diameter. Our experiments have shown so far that, with this probe, mass spectral information can be ascertained from molecules on a single bead provided that the attachment bond is somehow cleaved before measurement. The main advantage of this scheme is that many hundreds of beads can be assayed in a single measurement by rastering the probe beam across an array of target spheres (7). At the present time, for example, we have obtained spectral information at the rate of 1 beads/s using model systems. At this rate, it would clearly be possible to completely characterize rather large combinatorial libraries of a million members or more in just a few days. An important reason why these kinds of assays have not yet been achieved is that molecular desorption by energetic ion beams often leads to extensive fragmentation, thus complicating analysis (8). Moreover, all of the desorbed molecules must originate from the sample surface, a disadvantage in dealing with conventional polymeric solid supports where much of the action occurs inside the material. Here, we evaluate the prospects for using imaging TOF-SIMS to assay large numbers of compounds on beads or other supports in a short period of time. Our Intensity Intensity Intensity m/z m/z m/z Figure 2. Molecule-specific imaging. These are two 2- m polystyrene beads, each covered with a different peptide mimic with molecular weights 226 and 547 and placed on a silicon substrate. The top panel shows an image of all of the positive ions emitted from the field of view and the mass spectrum associated with that image. The remaining panels show mass spectra and images associated with the specific regions of space corresponding to the presence of one bead or the other. From the spectra, it is obvious which species is associated with a specific bead. (Sample courtesy of D. Wagner and M. Geysen.) evaluation begins with a description of how this type of MS differs from other types, and proceeds by showing examples of several preliminary experiments on single beads. We also focus on the goal of characterizing arrays of beads in parallel fashion so as to achieve the full potential of the combinatorial chemistry idea. EXPEIMENTAL TECHNIQUES Figure 1 shows a schematic diagram of an imaging TOF-SIMS spectrometer. Details are available elsewhere (9). The mass spectra are generated by ion bombardment using a pulsed liquid metal ion gun (LMIG). These sources consist of a sharp tungsten tip that is coated with liquid gallium. A field of 25 kev extracts SEPTEMBE 21 16(9) SPECTOSCOPY 15

3 I M A G I N G Intensity (a) (c) m/z Intensity Figure 3. Molecule-specific imaging in which the images are recorded by plotting the total intensity of ions within the indicated mass window as a function of position on the surface. The images were produced using (a) only the red channel of a standard GB 24-bit image at m/z 227, (b) only the green channel at m/z 548, and (c) by adding the two upper images together. The resulting picture keys a specific color to a specific mass range. (b) m/z Ga+ or In+ ions from the tip. With appropriate ion beam optics these ions may be focused onto the sample surface into a spot size of 5 nm in diameter. Metal lost at the tip is replenished by electrohydrodynamic flow, and the beams will persist for 1 h of operation. Pulsing is achieved by using apertures and deflection plates. By using fast electronic switching, we can easily achieve pulse widths of 1 ns with a repetition rate of 1, pulses/s. In addition to the LMIGbased probe s small size, it also produces an extremely bright source of ions. For our work, we had 1 na of ion current focused into a 1-nm spot, yielding a current density of 1 A/cm 2. It is also interesting to note that a 1-ns ion pulse contains 62 Ga + ions and that 1 4 molecules per layer are exposed to the beam. Once desorbed, the ionized molecules are extracted into a reflecting mirror TOF mass spectrometer, as shown in Figure 1. The extraction voltage is 6 kev and the TOF drift voltage of 2.5 kev is applied over a distance of 3 m. Hence, the flight time of an ion of m/z 8 is 1 s. These analyzers generally achieve quite high performance. For example, the transmission efficiency is on the order of 5%, and the mass resolution exceeds one part in 5. Moreover, with appropriate calibration, m/z can be determined to a few ppm accuracy, an important feature for composition studies. A limitation of this design involves the field of view of the analyzer and the magnitude of the deflection of the ion beam. Because of the magnitude of the voltages and the requisite scan speed, a 2 mm 2-mm raster pattern is the largest possible scan area for a primary ion beam energy of 25 kev. Moreover, the ions entering the TOF analyzer must maintain their time coherence, so ions emitted from a region larger than several square millimeters will not reach the detector. Hence, molecule-specific images are restricted to an area of this size. The instrumentation is equipped with a low-energy electron gun (not shown in Figure 1) for surface charge compensation purposes. On insulating materials such as our polystyrene beads, an imbalance of charged species emitted from the surface can result in the buildup of a surface potential. This potential can, in principle, be nearly as large as the energy of the primary ion beam and deflect secondary ions away from the TOF analyzer. To account for this phenomenon, the sample is flooded with a pulsed 3-eV electron beam that has a current density of a few microamperes per square centimeter. The timing is adjusted so that the beam strikes the target for 5 s during the time when the secondary ions are already in the drift tube. This procedure effectively neutralizes any charge buildup and allows the analysis to proceed in an unimpeded fashion. Imaging is achieved by moving the ion beam over a given area of the target and recording mass spectra on the fly. Figure 2 shows an example of this kind of image. Typically, a picture like this contains pixels and the Ga + ion beam strikes each pixel 1 times. At 1, mass spectra/s, the image is recorded in 64 s. This number of pulses keeps the Ga + ion beam dose within a regime that does not observably change the surface chemistry by beam damage, commonly referred to as the static SIMS limit. For the image in Figure 2, each pixel represents an area of 1 m 2 or 1 6 molecules per layer and is interrogated with a dose of Ga + ions. Only.6% of the surface sites then are actually sampled. The resulting data file contains information associated with 65,536 mass spectra. Although this number at first seems daunting, data compression techniques 16 SPECTOSCOPY 16(9) SEPTEMBE 21

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5 I M A G I N G fs 535 fs Figure 4. Molecular dynamics computer simulation of molecular desorption. actually make storage and retrieval quite straightforward. During imaging, position and flight time information is stored for each detected ion. This file can then be sorted quickly to provide images associated with a particular flight time or m/z value. An example of how the analysis proceeds is shown in Figures 2 and 3. The size of this data file is 1 MB, and the image processing is completed in a fraction of a second with a standard personal computer. Finally, some experiments use a pulsed laser beam to photoionize the desorbed neutral molecules (1). This methodology is incorporated in Figure 1 and is discussed briefly near the end of this section. For these experiments, the laser is fired 1 ns after the primary ion pulse. The sample voltage is also pulsed to eliminate spurious ions. The results presented in this work do not yet use this feature. ESULTS AND DISCUSSION The mechanism of molecular ion formation in SIMS is distinct from that observed with related forms of MS such as MALDI and electrospray MS (ESMS). The presence of energetic collisions from a 25-keV ion source generally induces more fragmentation than softer ionization schemes. The fact that molecules desorb mainly from surface layers produces special sample-handling issues that are not associated with techniques that deal with bulk materials. Here, we evaluate the prospects for overcoming these difficulties and detail how to exploit the high speed and spatial resolution associated with TOF-SIMS measurements. Mechanisms of molecular desorption. It is of interest to consider some of the mechanisms by which surface molecules are desorbed by energetic ion beams. The basic idea is that the incident projectile sets in motion a large number of atoms near the sample surface (11). Because the attractive forces that hold the solid together are generally in the range of 3 6 ev, eventually a few particles escape and are detected. Extensive local disruption occurs near the point of impact of the primary ion, and it is remarkable that intact molecular desorption occurs so readily. The details of this disruption have been modeled by molecular dynamics computer simulations (12, 13). These calculations provide an atomic-level view of the motion that leads to desorption. Figure 4 shows the results of a typical calculation for 5-eV Ar + ion bombardment of a layer of C 2 H 3 on a platinum crystal (12). The results indicate that the desorption event is finished in 5 fs and that the disruption area extends to 1 atoms ( 25 Å) away from the impact point. We observe fragment species and parent molecular species in these calculations. The desorption is induced by atoms moving at just a few electron volts after the primary ion energy has been transferred to many more slowly moving atoms. Very similar pictures have been reported using more energetic ions such as the ones employed in this section. Note that these moving atoms, generally referred to as the collision cascade, cause desorption of molecules from an area that is comparable to or smaller than the size of the ion beam probe. Ionization can occur by direct collisional excitation or by cationization with protons or other charged ions in the desorption flux. Covalently linked surface molecules. Our goal is to interrogate an array of beads and acquire mass spectral information from each bead in parallel fashion. This goal seems feasible enough because the SIMS technique is known to desorb molecules directly from the surface of a wide variety of materials and with high lateral resolution. For molecules synthesized on beads, however, uninterpretable mass spectra usually ensue using this strategy. Figure 5 shows an example of this. Here stearic acid, selected as a model compound, was attached to a 5- m bead using the acid-sensitive Sasrin linker and then bombarded with the Ga + ion beam. The mass spectrum consists entirely of low-mass fragment ions, which yield little structural information about any stearic acid that is desorbed. We know, however, that stearic acid itself, when prepared as a thin film, yields very clean mass spectra with peaks at (M H) + and (M H 2 O H) + where M is the molecular weight of stearic acid. In fact, it would be feasible to delink the stearic acid from the bead, 18 SPECTOSCOPY 16(9) SEPTEMBE 21

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7 I M A G I N G (a) Intensity (counts) (b) Intensity (counts) (c) Intensity (counts) St prepare a thin film, and complete the assay. This is essentially the strategy employed by MALDI or ESMS in characterizing molecules that are synthesized on beads. Although this procedure can work, lateral resolution is lost because molecules are not confined to specific beads, and the parallel aspects of the assay cannot be carried out. It is interesting to briefly examine the mechanisms that cause the linker to influence the SIMS spectrum. Computer simulations that use a model system of pentylidyne adsorbed on platinum have been performed in which the strength of the covalent interaction between the adsorbate and the substrate was changed arbitrarily (14). The results show that for Figure 5. Mass spectra of stearic acid under various conditions. Spectra obtained (a) from stearic acid attached to polystyrene spheres using the Sasrin linker, (b) after exposing the sample in (a) to a gas phase mixture of TFA and methyl chloride for 3 min, and (c) using the ink linker. O O St O St N 2 St St St weak interactions, the collision cascade can successfully eject intact molecules with minimal fragmentation. For stronger interactions, however, smaller fragment species created by energetic collisions can move laterally and collide with adsorbate molecules. These collisions tend to break the molecule near the bonding point on the surface, causing ejection of the top molecular piece. This mechanism is illustrated schematically in Figure 6. Hence, both experimental observations and computer simulations suggest that the link between the library member and the bead needs to be severed before mass spectral assay. To achieve this condition, we have developed a protocol for clipping the covalent surface attachment while leaving the target molecule resting in place on the bead (6). This protocol involves placing a single bead or array of beads into an enclosed chamber saturated with trifluoroacetic acid (TFA) and methylene chloride vapors from a 15% TFA in methylene chloride solution. A 3 3 min exposure is sufficient to cleave the molecule from the bead. The progress of the reaction can be monitored by the observation of a color change from off-white to purple on the beads themselves. Once the cleavage reaction is complete, the array can be inserted directly into the TOF-SIMS for analysis. The effect of this protocol on the SIMS data is shown in Figure 5, where spectra for stearic acid linked to a polystyrene bead by either Sasrin, ink, or Wang linkers and vapor-phase TFA clipped are shown. Note the appearance of the (M H) + ion at m/z 285 and the (M H H 2 O) + ion at m/z 268 for the Sasrin- and Wang-linked beads, and the appearance of the stearamide molecular ion for the ink-linked beads. In the latter case, the point of attachment is through an amine functionality. In addition to enhancing the information content of the SIMS spectra, the gasphase TFA treatment preserves the spatial configuration of the molecules. The images shown in Figures 2 and 3, for example, were treated with TFA vapor. From the color encoding scheme, it is obvious that there is no movement of molecules either off the bead or onto a neighboring bead. This very clear situation is not always observed, however. As Figure 7 shows, exposure to excessive TFA vapor can cause some molecules to be washed off and onto the holder. For this case, the tripeptide Val-Tyr-Val was bound to a 6- m sphere via the Sasrin linker. After TFA exposure the Y 2 2 peptide fragment at m/z 281 was imaged with the bead present and after removal of the bead. Although the molecule is still found to be localized on the target, it is also found in a halolike residue on the substrate itself. Because clipping can produce artifacts such as these, more research is needed to find self-cleavable linking agents or alternative means of releasing target molecules from the bead without changing their location. The use of photosensitive linkers represents one possible approach of this type. Compound identification with SIMS. A critical issue is whether MS in general or SIMS in particular has enough molecular 2 SPECTOSCOPY 16(9) SEPTEMBE 21

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9 I M A G I N G (a) (a) (b) (b) Figure 6. Molecular dynamics simulation of ion-bombarded C 5 H 9 on platinum{111} in which (a) the molecules are assumed to interact with the surface with a binding energy of 2 ev (chemisorption) and (b) the interaction energy is only.1 ev (physisorption). Note the difference in fragmentation when desorption is initiated by energetic reaction products moving laterally along the surface (14). Figure 7. (a) Total ion image of a 6- m bead bound with Val-Tyr-Val; (b) pixel image of m/z 281, with the bead removed from the sample. specificity to correctly determine the chemical composition of each member of a large combinatorial library. From molecular weight information alone this goal is certainly problematic. For example, there are members of a pentapeptide library synthesized from the 2 naturally occurring amino acids. Their molecular weights extend from m/z 35 to 125. From SIMS measurements, mass accuracy of about.1 amu and mass resolution of 1 part in 5 or better allow separation of all nonisobaric peptides except those involving leucine and isoleucine. These latter units may be differentiated by incorporating an 15 N label into one. Still, many mass redundancies are possible with compositionally different peptides such as Phe-Trp-Trp-Trp- Asn and Phe-Trp-Trp-Tyr-His (837.36) or permutationally different peptides such as Phe-Trp-Trp-Tyr-His and Phe-Trp- Tyr-Trp-His. To distinguish between library members of exactly the same molecular weight, it is possible in some cases to use the fragment ions that are always observable in the spectra. For peptides, a complete set of fragment ions is generally observable for small peptides (15). It is also feasible to use tandem MS by intentionally fragmenting the parent ion with a collision gas and performing MS on the reaction products (16). This strategy has only rarely been used with SIMS and has yet to be tested with combinatorial library assays. Similar issues exist when examining nonpeptide libraries. In a recent study, for example, the quasimolecular ion of an angiotensin II receptor antagonist was observed by SIMS, MALDI, and ESMS (3). The fragmentation pattern with SIMS, although distinct from that observed by the other methods, provided additional insight into the molecular structure. Tags and codes. An obvious potential advantage of MS methods is that they offer the possibility of direct structural identification without the use of additional tagging or coding strategies. These strategies are clearly successful, but they add more reaction steps to the process. Moreover, although reading of the tag can provide a history of the reaction chemistry performed on the bead, it does not yield information about the success or failure of that reaction chemistry. In some cases, however, the use of MS-readable tags or codes may be necessary for a variety of reasons. For example, the molecular ion peak may simply not be visible by SIMS, or there may be sources of impurity species that make direct spectral analysis impossible. At least one approach to solving this problem has been demonstrated by Geysen and co-workers (17). In their example, they have embedded a known number of as many as five isotopic labels into the molecule of interest. Hence, any major peak associated with the target molecule will have as many as five additional mass peaks associated with them. The example in Figure 3 was constructed using this type of encoding pattern. Although designed for ESMS studies, the strategy appears to yield very similar spectra us- 22 SPECTOSCOPY 16(9) SEPTEMBE 21

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11 I M A G I N G (a) (b) m/z SPECTOSCOPY 16(9) SEPTEMBE m/z Figure 8. Comparison of (a) SIMS and (b) ESMS for an isotopically encoded peptide mimic. For (a), the molecule was extracted from the bead with TFA, deposited on a gold substrate, and analyzed as a dried thin film. For (b), the extract was injected directly into the ionization source of the spectrometer. Note that the intensity ratios of the peaks in a given cluster are very similar but that the amount of (M Na) + at m/z is much greater with SIMS. (ESMS courtesy of D. Wagner, Glaxo Wellcome [esearch Triangle Park, NC].) ing SIMS. One significant difference, however, involves the formation of quasimolecular ions with Na + or K + rather than with H +. Because solid-phase samples are often formed in the presence of a salt, these cations can easily attach to desorbing molecules, thereby increasing their apparent molecular weight. With ESMS, however, the samples can be purified using liquid chromatography, eliminating this complication. An example of this effect is shown in Figure 8, which compares the SIMS and ESMS of the same isotopically encoded compound synthesized on bead and extracted with TFA. Note that although one group of peaks is shifted by m/z 22, the relative intensities of the isotopic components are identical. This result further supports the use of tags to overcome complications in the interpretation of mass spectra and to enhance general applicability. It will be interesting to see whether MS-readable tag molecules can be developed to further expand options. Some attempts have already been successful in this direction for MALDI analysis of bead-bound DNA fragments. Arraying of beads for parallel assay. The long-term payoff for imaging MS in combinatorial library research focuses on the possible characterization of a large number of beads in parallel fashion. There are many envisionable scenarios that could lead to ultrahigh-throughput screening solutions, but perhaps the most direct modality is illustrated in Figure 9. In this instance, 5- m beads are arrayed into a specially designed metallic disk that is fabricated with holes of appropriate sizes. This disk could be chemically assayed with imaging TOF-SIMS on one side and screened against a variety of receptors on the other side. Bioactivity can be checked using any number of traditional handles including autoradiography or fluorescence microscopy. Information from the two sides of the disk is then correlated to acquire chemical structure activity relationships. Protocols necessary to accomplish this scenario are emerging rapidly. For example, we have fabricated a prototype beadholder of the type described above using a double-sided photolithographic technique. Positive and negative masks are cast onto the surfaces of a copper disk using an appropriate photoresist. The negative mask is then electroplated with nickel so the exposed copper regions on the positive mask can be etched with chromic acid. Once the photoresist is stripped from the metal substrate it is gold-plated to improve chemical robustness. Beads are then loaded directly into the array by forcing a liquid slurry through the holes. Figure 1 shows an optical picture of this array and a schematic cross-sectional diagram. To complete a chemical assay using imaging TOF-SIMS, the array shown in Figure 1 is filled with beads that are created in a split-and-mix combinatorial chemistry synthesis. Our chip was constructed for 5- m spheres and contains as many as 1, beads/cm 2. Note that we have chosen to use fairly small spheres rather than the more standard 2- m beads in an attempt to create as high a density as possible. As noted earlier, the field of view of the mass spectrometer is approximately 2 mm 2 mm, implying that as many as 4 beads can be assayed in one TOF-SIMS image. A typical example, shown in Figure 11, uses the stearic acid model system described earlier. In this picture, the red color denotes the presence of stearic acid, and the blue color denotes the presence of ions associated with the arraying disk. Each picture is displayed using pixels with 1 Ga + ion pulses of 1 ns duration applied to each pixel at the rate of 1, pulses/s. Hence, each image requires 1 min to acquire. Considering that the beads occupy slightly less than 2% of the surface area for this configuration, the Ga + ion beam interrogates each bead for 65 ms, shown in Figure 11a, and 32 ms, shown in Figure 11b. The assay of the entire array of 1, beads could be completed in less than an hour by moving the sample into an appropriate section of 25 tiled 2 mm 2 mm images. Sensitivity issues. Ion beam induced desorption occurs primarily from the surface layer of the sample. For polystyrene spheres, the solid-phase synthesis procedure allows activation of sites throughout the bulk of the material. A 2- m bead, for example, is capable of carrying as much as a few nanomoles of the desired compound. Yet, even if all sites are occupied, only a few femtomoles of material are present on the surface and amenable for SIMS analysis. These simple observawww.spectroscopyonline.com

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13 I M A G I N G Figure 9. Schematic diagram of arraying strategy. Figure 1. Optical micrograph of bead array, and a schematic diagram showing a cross section of one of the holes. tions suggest that the polymer surface must be kept rather clean because impurities can effectively cover the molecule of interest, rendering it invisible. Moreover, hydrophobic or coulombic interactions that occur on the surface could potentially force surface molecules into the bulk and decrease the signal. Sensitivity is obviously a key issue if this strategy is to become a practical screening tool. It is especially important if the technique is being used with codes or tags that may only be present at the 1 1% level. So far, the results suggest that the limit of detection is on the order of a few femtomoles for peptides (6) and a bit lower for an angiotensin II antagonist (3). Model compounds such as (a) (b) Figure 11. Molecule-specific TOF-SIMS images of an array of stearic acid beads. The Ga + ion beam interrogated each bead for (a) 65 ms and (b) 32 ms. stearic acid exhibit detection limits at least an order of magnitude lower, presumably because of the high ionization efficiency of these types of systems. Although these limits are generally satisfactory, there are many possible schemes for increasing sensitivity further. One possibility that has been exploited by many groups involves the use of an intense, pulsed UV laser to photoionize any neutral molecules that have desorbed from the surface (1). This strategy is attractive in principle because we know that more than 99% of the desorbed flux is, in fact, neutral. Anecdotal reports of examples of systems indicate that sensitivity improvements of an order of magnitude have been observed. Because of molecular photodissociation, however, the method is not yet general enough to be applied to a large set of similar molecules. Another possibility involves the use of a matrix molecule to enhance the SIMS yield. This scheme follows the observations leading to enhanced molecular ion emission by MALDI and appears to be successful for some systems. Preliminary experiments in which a matrix is added to the bead surface are so far inconclusive, but work is continuing (18). Finally, molecular ion yields are found to be enhanced by the use of cluster ion beam sources (19). Apparently, the collection of incident particles is more effective at initiating the collision cascade than a single particle of equivalent mass, particularly with light substrates such as polystyrene. Some lateral resolution has been achieved using SF 6 as the projectile, and yield enhancements of at least 1-fold have been observed. It will be interesting to see if this advance can further improve detection limits, especially if imaging cluster sources can be found. 26 SPECTOSCOPY 16(9) SEPTEMBE 21 CONCLUSIONS AND POSPECTS Here, we have attempted to show how the marriage of MS, nanofabrication, and imaging can lead to new ultrahighthroughput screening opportunities for combinatorial chemistry research. Although the preliminary experiments that have been completed appear promising, it is obvious that a number of technical hurdles remain to be overcome before this method can be reliably implemented. The biggest problem involves the fragmentation of desorbing molecules by the Ga + ion probe. Many solutions to this problem present themselves for future research, including development of embedded isotope labels or tags and the incorporation of special matrices that enhance the molecular ion yield. Another issue involves the selection of the most appropriate configuration for chemical and bioassay using a sample holder similar to the prototype described here. So far, large libraries have not been examined, and it is unclear whether mass redundancies and other interferences will be serious deterrents. The possibility of creating a spatially encoded array of beads that is directly amenable to bioassay, however, appears to be eminently feasible. In summary, we have demonstrated that read rates of greater than 1 beads/s are possible using imaging TOF-SIMS under the right set of experimental parameters. With this capability, it should ultimately be possible to achieve a great deal of information about the chemical reactivity of large combinatorial libraries that has not been possible previously. By covwww.spectroscopyonline.com

14 I M A G I N G ering a larger domain of molecular composition, many of the drug discovery concepts created by the combinatorial approach may be fully realized. EFEENCES (1).S. Youngquist, G.. Fuentes, M.P. Lacey, and T.J. Keough, J. Am. Chem. Soc. 8, 77 (1994). (2) B.J. Egner, G.J. Langley, and M.J. Bradley, J. Org. Chem. 6, 2652 (1995). (3) C.L. Brummel, J.C. Vickerman, S. A. Carr, M.E. Hemling, G.D. oberts, W. Johnson, J. Weinstock, D. Gaitanopoulos, S.J. Benkovic, and N. Winograd, Anal. Chem. 68, 237 (1996). (4) Y.-H. Chu, Y.M. Dunayevskiy, D.P. Kirby, P. Vouros, and B.L. Karger, J. Am. Chem. Soc. 118 (33), (1996). (5) P.K. Jensen, L. Pasa-Toli, G. A. Anderson, J. A. Horner, M.S. Lipton, J.E. Bruce, and.d. Smith, Anal. Chem. 71, 276 (1999). (6) C.L. Brummel, I.N.W. Lee, Y. Zhou, S.J. Benkovic, and N. Winograd, Science, 264, 399 (1994). (7).M. Braun, M.L. Pacholski, A. Beyder, and N. Winograd, Secondary Ion Mass Spectrometry (SIMS XI), G. Gillen,. Lareau, J. Bennet, and F. Stevie, Eds. (Wiley, New York, 1998), p. 89. (8) Secondary Ion Mass Spectrometry, Principles and Applications, J.C. Vickerman, A. Brown, and N.M. eed, Eds. (Oxford University Press, 1989). (9).M. Braun, P. Blenkinsopp, S.J. Mullock, C. Corlett, K.F. Willey, J.C. Vickerman, and N. Winograd, apid Commun. Mass Spec. 12, (1998). (1) K.F. Willey, V. Vorsa,.M. Braun, and N. Winograd, apid Commun. Mass Spec. 12, (1998). (11) Sputtering by Particle Bombardment III,. Behrish and K. Wittmaack (Springer- Verlag, Berlin, 1991). (12).S. Taylor and B.J. Garrison, J. Am. Chem. Soc. 116, 4465 (1994). (13). Chatterjee, Z. Postawa, N. Winograd, and B.J. Garrison, J. Phys. Chem. 13, 151 (1999). (14).S. Taylor and B.J. Garrison, Int. J. Mass Spec. Ion Processes 143, 225 (1995). (15) K. Biemann and S. A. Martin, Mass Spectrom. ev. 6, 1 (1987). (16) V.M. Doroshenko and.j. Cotter, Anal. Chem. 68, (1996). (17) H.M. Geysen, C.D. Wagner, W.M. Bodnar, C.J. Markworth, G.J. Parke, F.J. Schoenen, D.S. Wagner, and D.S. Kinder, Chem. Biol. 3, 679 (1996). (18) K.J. Wu, T.F. Fister, and.w. Odom, 12th Annual SIMS Workshop, Gaithersburg, MD, April 25 29, (19) G. Gillen and S. oberson, apid Comm. Mass Spec. 12, (1998). Nicholas Winograd is Evan Hugh Professor in the department of chemistry at Penn State University, 184 MI Building, University Park, PA He may be contacted by telephone at (814) 863-1, by fax at (814) , and by at nxw@psu.edu. obert M. Braun is a senior laboratory scientist at Evans PHI Laboratory, 659 Flying Cloud Drive, Eden Prairie, MN He may be contacted by phone at (952) , by fax at (952) , or by at rbraun@phi.com. Circle 17 SEPTEMBE 21 16(9) SPECTOSCOPY 27