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dard. The least amount of product was generated in three vials that contained the best inhibitory components.

D.Surface Enhanced Affinity Capture and Probe Affinity Mass Spectrometry

One of the advantages of matrix-assisted laser desorption ionization (MALDI) is its sensitivity. As described above, MALDI can be extremely useful for the identification of solid phase combinatorial library components and even sequencing of peptide libraries using a single bead. Therefore, it is also a useful technique for screening combinatorial libraries where sample quantities do not permit the application of immunoaffinity HPLC-MS or ultrafiltration techniques with high flow rates, limited sensitivity, and significant consumption of receptor. Specific examples of applying MALDI to the identification of high-affinity ligands have been published by Hutchens and Yip (125) with their use of surface enhanced affinity capture (SEAC) and others. (128,129). In this approach, a receptor molecule is immobilized on agarose beads, and the analyte is captured directly out of solution. For this first published example, lactoferrin was detected from preterm infant urine by the addition of prepared beads to the urine solution. After removing and washing, the beads were placed on the MALDI target and the ligands detected. Other examples have shown that it is possible to directly detect an anti-monoclonal antibody to cytochrome c from cytochrome c immunoaffinity column (IAC) material. These experiments are useful for concentrating the analyte and providing affinity screening but some interference with the capture medium e.g., the IAC material or the agarose beads, has been observed.

In an alternative approach, Brockman et al. (121) have worked around these problems by immobilizing their analytes directly onto the surface of the MALDI probe (141). Using disposable MALDI probe tips coated with gold, they generated self-assembled monolayers that were then used to immobilize antibodies directly onto the tips. Figure 13 shows how the dextran based probe affinity mass spectrometry (PAMS) surface can screen for binding ligands. The binding molecules in this case were anti-γ INF antibodies that were attached to the dextran. It was possible to overcome the problem of nonspecific electrostatic interactions between γ INF and the immobilized anti-γ INF antibodies by increasing the number of binding sites through oxidation of the dextran molecules and subsequent immobilization of the anti-γ INF antibodies, thus avoiding generation of carboxyl groups. This approach may not be applicable to all kinds of analytes but, if both the immobilization chemistry and the

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Figure 13 Mechanistic illustration of the probe-based affinity separation. (Reprinted from Ref. 121.)

analyte recognition work, it can be very useful for trace analysis and analyte enrichment on the MALDI target.

Youngquist et al. (63) have used color indicators for the identification of beads carrying active components. Bead-bound combinatorial libraries were subjected to a selection procedure that left dark blue stains on those peptide-

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containing beads that were binding to the selected antibody. Eight stained and five nonstained beads were chosen from the affinity screening experiment; then the bound peptides were digested and analyzed by MALDI as described in Sec. II.B. The known recognition sequence of the antibody was found in six of the peptide samples isolated from the eight stained beads. Fewer matches were obtained for the two samples derived from the other two stained beads and no matches were found for samples originating from unstained beads.

E.Bioaffinity Characterization–Mass Spectrometry

A method for screening receptor–ligand interactions has been introduced by Smith et al. and Ganem et al. (107,142–146) with their approach of bioaffinity characterization–mass spectrometry (BAC-MS). The method relies on the gas phase interactions present between the receptor and the binding ligands when using ESI. Both ESI and MALDI are soft ionization techniques that produce stable gas phase ions of large molecules without fragmentation. It is also possible to see noncovalent complexes in the mass spectrometer using these techniques (147), but recent research has focused on the specificity of the monitored noncovalent or electrostatic interactions that occur in the gas phase and many questions have remained open as to whether solution and gas phase complexes should be compared. Nevertheless, in an interesting experiment, a receptor and components from a combinatorial library were incubated in solution and after equilibration the liquid was electrosprayed directly into an FTICR high-resolution mass spectrometer. The FTICR instrument allowed the accumulation of selected ions or ion complexes in its ion trap and thereby provided a separation step. The noncovalently complexed molecules were then dissociated by applying additional energy. The remaining ligands were retained in the ion trap and were subjected to further MS/MS studies for characterization. Figure 14 shows the principle of BAC-MS. Bradykinin and bovine ubiquitin were used as model components.

Additional information on library screening can be found elsewhere in this volume (Chap. 8).

III.FUTURE DIRECTIONS FOR THE APPLICATION OF MASS SPECTROMETRY IN COMBINATORIAL CHEMISTRY

The most important reason for the success of combinatorial chemistry is the much greater efficiency when synthesizing, analyzing, and screening compo-

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Figure 14 A conceptual representation of the BAC-MS technique. (a) The mixture solution is ionized by electrospray ionization and the complex of interest is selectively accumulated in the FTICR trap. (b) The noncovalent complex is then ‘‘heated’’ to liberate the affinity ligand species. (c) The ligand ions are retained for further CAD studies to present structural information. ‘‘T’’ represents the target biomolecules, and ‘‘L’’ represents ligands that form high-binding-affinity-specific noncovalent complexes with the target molecules. Circles (solid and empty) represent ions from other molecules present in the mixture. (Reprinted from Ref. 142.)

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nents. Combinatorial chemistry with its possibility of automation has been found to increase sample throughput per person by a factor of 8 or more since the early 1990s (7) and robotic high-throughput systems are mainly responsible for this dramatic increase (148–150). For even higher sample throughput it is desirable to use a setup for automated synthesis directly together with MS. The analysis can be extended to include other characterization and screening methods like ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy (56,151). Zuckerman et al. (79) used an equimolar peptide mixtures (EPM) synthesizer to generate libraries of up to 36 individual peptides or peptide mixtures. Each well was screened in an enzyme-linked immunosorbent assay (ELISA) and the identified binding ligands were then characterized in a glycerol matrix by secondary ion MS. Boutin et al. (78) have constructed a Zymark-based robot to synthesize their combinatorial libraries with subsequent analysis by MS, CE, and NMR.

For higher resolving power and smaller sample quantities it may be desirable to use a capillary electrochromatography (CEC) system as has been done by Zweigenbaum et al. (44) for their separation of a mixture of steroids. The CEC system was coupled on-line to an ion trap mass spectrometer. CEC is especially useful for the separation of isomers.

Other examples of future developments in combinatorial chemistry related to MS include the use of animals within pharmacokinetic studies (152,153). The coadministration of several components to one animal could dramatically speed up the discovery process for metabolites (154,155). Also, automated combinatorial degradation profiling has been shown to add another dimension to the automated processes of mixture synthesis and analysis (98,156,157).

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