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Vouros and Dunayevskiy and others have utilized free solution or affinity CE methods combined (interfaced) with MS detection for searching libraries, as described below (18). However, CE or affinity CE is nothing but a separation technique, and it cannot and never will identify structures, especially when one is unsure of the contents of a given library. CE is a solution-based approach, though capillary gel electrophoresis (CGE) does use a polymeric gel in the capillary, which separates on the basis of differences in mass-to-charge or charge- to-mass ratios. This almost sounds like the basis of separations in MS. The reader must be referred to the available texts or review articles that describe how CE works, instrumental requirements, and the foundations or fundamentals of its separating abilities (36).

There are numerous variations on free solution CE (FSCE), such as micellar electrokinetic capillary chromatography (MECC or MEKC), where a moving, pseudostationary phase is added to the CE buffer, and secondary chemical equilibria or interactions ensue that effect separations of even neutral compounds, as well as ionic analytes. However, in general, CE utilizes truly homogeneous, solution phase separation approaches, without a stationary (permanent, fixed) phase, making it perhaps ideally suited for molecular recognition in searching combinatorial libraries.

In affinity CE (ACE), one adds to the running buffer or to the sample, the recognition element or ligand, and then looks for changes in mobility patterns of individual members of the sample (mobility shifts, as in flat-bed electrophoresis). This has been described in many publications, without searching combinatorial libraries, as a method to identify antibody–antigen partners, active antibodies, and active antigens. Thus, ACE is really a variation on CE, by virtue of adding something to the running buffer or, more often, to the sample that will then provide different mobility tendencies and migration patterns (mobility shifts). The two different electropherograms generated, with and without added ligand or receptor, provide differences that can be utilized to identify active partners, analogous to difference chromatography, used for many years in HPLC (19). A variation of affinity CE, if you will, permits the detection of low levels of enzymes by placing the enzyme substrate into the running CE buffer, something termed enzyme-modulated microanalysis (EMMA).

At the same time, there are some limitations in utilizing CE for library search routines. CE utilizes very small amounts of sample, nanoliter volumes, and thus very low levels of possible recognition elements. Because detection is usually through the capillary, except for MS, detection limits are very high (poor), making this a generally insensitive technique for trace analysis. There are some recent improvements in detector cell design, bubble cells, Z cells,

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rectangular capillaries, and so forth that have lowered detection limits for many analytes in CE (13,14). Also, since ultraviolet detection (UV)/fluorescence detection (FL)/electrochemical detection (EC) methods provide little, if any, actual structural information about a specific CE peak, applications of CE for combinatorial library searching really require interfacing with MS detection (37). MS will often provide much lower detection limits for library components, and (most importantly) it can often provide structural information and even absolute identification (38).

CE also has the ability of providing improved resolutions for similar structures, such as peptides, which may be difficult or impossible to separate by any and all HPLC methods (39). Because it moves analytes and the buffer by electroosmotic flow (EOF) rather than by pressurized flow delivery as in HPLC, individual peaks tend to be much narrower, sharper, symmetrical, and of higher plate counts (efficiency, N (number of theoretical plates or plate count for peak)). This also leads to improved resolutions of nearly identical compounds, assuming that they have some differences in mass-to-charge ratios or can interact with buffer components differently, as with their ligands or receptors.

It would therefore appear at the outset that CE may provide the best approaches for identifying lead, active compounds from large libraries of possible targets. There are, of course, some limitations in using current, solution phase methodologies. Primary among these possible or real limitations is the idea that activity of a given pool is dependent on the cumulative activity of all compounds present. It is not always guaranteed that one will locate the true, optimally active compound. One may have missed the best candidate of all, depending on how that library was synthesized and its final contents. It is also quite possible that in using a library of many compounds, recognition may be a result of synergistic or summary effects, rather than the true effect of just a single, individual compound. This is very common, and often the larger the library, the more likely synergism may play a role. It is also possible, at times, that finding synergistic compounds, more potent when administered together, is a desirable outcome. Though perhaps more difficult to sell to the Food and Drug Administration (FDA) for final, investigational new drug (IND) approval, it has been done many times.

This fault runs counter to the one just mentioned, which suggests that to find the true optimum compound one should be using larger and larger libraries, which only makes the possibility of synergism more and more likely. What a conundrum. These conflicting demands are not necessarily easily resolved, other than to utilize individual compounds for testing. However, that only defeats the goal of being able to screen larger and larger combination

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libraries in shorter and shorter times with less and less money and manpower. Thus, while using purely solution phase screening strategies possible in CEMS seems ideal, clearly certain loopholes and pitfalls remain. Nothing in life or science is perfect, and we are surrounded by gray areas, where we would prefer black or white. There may be no simple way to resolve this lingering problem.

II. AFFINITY CAPILLARY ELECTROPHORESIS

This section is devoted to a description and summary of those literature reports wherein some form of affinity recognition has been described. None of these reports actually utilized a combinatorial library for such recognition; those applications follow. However, in order to appreciate how and why ACE plays such a large role in using CE-MS for combinatorial library searching, one must begin with the more general and simpler use of affinity recognition in CE. We describe here the use of some type of biorecognition element, be that antigen, antibody, receptor proteins, binding drug, or ligand, that interacts with a partner to provide altered electrophoretic migration tendencies and thus mobility differences in CE. As a result of temporary, noncovalent association or binding of the ligand to its receptor molecule, it experiences a real difference in its normal, free migration tendency and appearance in the final electropherogram. One can then imagine generating two difference electropherograms: that for the ligand alone in the absence of its receptor, and that in the presence of the receptor, where some degree of affinity or binding will occur. Depending on the migration tendency of the receptor molecule relative to that of the ligand, which is usually different, one should observe a mobility shift of the ligand. The degree of this mobility shift is directly related to the affinity constant of the ligand for its receptor; the dissociation constant of the complex; the nature of the buffer medium and its effect on complex mobility; differences in mass-to-charge ratios of the ligand, receptor, and ligand–receptor complex; and perhaps other CE conditions. In order to make use of affinity recognition in CE or ACE, one must have real, discernible differences in the mobilities of the free ligand vs. its ligand–receptor situation.

We know of no specific review articles that discuss only affinity CE, though there is a very recent review that describes the use of CE for antibody analysis, often with applications that utilize antibody–antigen recognition called immunoaffinity or affinity CE, as above (40). At times, recent texts also contain sections that discuss affinity recognition in CE, such as that by Righetti (10). Because most applications of CE for combinatorial library searching

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have involved some type of biological recognition, as opposed to molecular imprinting (recognition) methods, becoming more familiar with ACE and its variations can only help us to understand and appreciate why ACE-MS techniques may be quite successful for future library searching.

We will therefore start by describing some recent applications of affinity recognition in CE and what these teach us about utilizing their principles and practical approaches for eventual application to combinatorial libraries. There are far more papers that utilize some form of ACE, but far less that then apply those methods for combinatorial library searching. Although there are some reports on the use of CE for general, group separations of compounds in larger combinatorial libraries, it does not appear that CE alone will provide significant advantages in discerning the contents and structures of individual members of a larger library. Thus, we have entirely emphasized here only those reports that utilized CE for some type of affinity or immunoaffinity recognition of a specific antigenic species, whether that was a small or large molecule. While most such reports have utilized small antigens in ACE, it is entirely possible that larger protein libraries will also be successfully searched using ACE methods.

In recent years, ACE has been used to study ligand–receptor interactions and determine the binding constants of formed complexes (41–43). Some typical applications include enzyme–inhibitor, DNA–peptide, protein–sugar, pep- tide–drug, and antibody–antigen (Ab–Ag), etc. We will focus on Ab–Ag interactions.

The adsorption of proteins to the inner wall of the capillary is a major consideration in ACE assays (44,45). The interaction can lead to band broadening, irreproducible migration times, low resolution, and low recovery of the protein. Various approaches to control the problem include chemical modification to the inner surface of the capillary, choosing a proper buffer species and pH, and the use of buffer additives to reduce the protein interaction with the capillary wall. The buffer additive has to be cautiously chosen so as not to participate in the interaction between Ag and Ab.

Ab–Ag interactions and complexation kinetics vary for different systems (42). Some possible CE patterns are shown in Fig. 1 for a receptor– ligand system. For a fast-interaction rate system, each protein molecule spends the same time forming a complex(es) with the Ab, and although the migration time changes, the peak shape does not. Of course, it is also possible that several Ab–Ag complexes may form in a system-dependent way which can only complicate the final CE analysis. For a slowly interacting system, some protein molecules spend more time forming a complex with the Ab than the others, which leads to broad peaks, or a disappearance of the peak altogether, or even

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Figure 1 Schematic diagram of some of the possible interaction patterns in affinity CE with hypothetical, homogeneous receptor–ligand systems characterized by different reaction kinetics. Ref. is a noninteracting component, whereas the other peak represents a molecule interacting quickly (second panel from top) or more slowly (lower panels) with ligands of lesser electrophoretic mobility, which are present in the electrophoresis buffer (42). (Reproduced with permission from the copyright holder, Elsevier Science Publishers and Journal of Chromatography.)

separate peaks for complexed and uncomplexed proteins. As the assay protocols are different for slow and fast kinetic systems, some initial experimentation needs to be performed to determine the exact kinetics prevalent in a given situation. Experiments similar to those in Fig. 1 can be performed, and the kinetics can be determined based on the observed peak shape(s) (42).

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In a system with fast kinetics, buffers containing different concentrations of Ag or Ab are prepared, and the sample having a fixed concentration of Ab or Ag is injected into the capillary. Scatchard analysis of the change in migration time of the Ab or Ag as a function of the concentrations of Ag or Ab in the buffer can then determine the binding constant.

In a system with slow kinetics, the Ag and Ab need to be preincubated before injection into the capillary (45,46). Fixed concentrations of Ag or Ab are then incubated with different concentrations of Ab or Ag. The quantity of free Ab or Ag can be determined by the peak area using a calibration plot. Scatchard analysis is made by plotting the total amount of Ab or Ag vs. bound Ab or Ag. For the intermediate kinetics system, separation conditions, such as applied voltage, length of the capillary, pH, and other factors, can be changed so that the system can be analyzed using one of the final experimental protocols.

III.SEPARATION AND DETECTION OF ANTIBODIES, ANTIGENS, AND ANTIBODY–ANTIGEN COMPLEXES BY HPCE METHODS

Reports on the separation of Abs by HPCE have mainly included the use of capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), with much less being reported by CGE methods (40). The separation mechanism of CZE is based on electrophoretic mobilities of the sample components, which are affected by solvent characteristics, including pH, ionic strength, and viscosity. The separation in CIEF is based on differences in isoelectric points (pI ). Most work on Ab separations by CE has been used to separate a monoclonal Ab because its homogeneity when compared with a polyclonal Ab often leads to a much higher specificity. This will also result in a higher sensitivity for the final immunoaffinity process as there will be a single peak or species detected, along with an improved possibility to develop simple and sensitive assay methods. Monoclonal Abs produced by hybridoma or genetic engineering techniques have often displayed a certain degree of microheterogeneity when analyzed by CE (47–56). This microheterogeneity may be caused by differences in glycosylation, variations in protein sequences, posttranslational modifications, improper folding, and other factors (57).

An Ab will interact with its Ag and form the usual Ab–Ag complexes. It is easy to see that if a polyclonal Ab were used for recognition with its Ag, the final electropherogram of the mixture could become quite complicated. For the above reasons, Nielsen et al. used the monoclonal Ab specific for

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human growth hormone (hGH) to study the CZE separation of Ag, Ab, and the various possible complexes (58). Theoretically, if the monoclonal Ab were used, only two types of complexes would be formed—those corresponding to reaction at the one or two Ag binding sites of each Ab molecule. Figure 2 illustrates a series of CZE electropherograms for immunoglobulin (IgG), hGH, and various mixtures containing an excess of IgG (Ab). This work demonstrated the ability of CZE to separate the Ag and Ab complexes from excess Ab and/or free Ag. In Fig. 2, it is apparent that incomplete resolution of all possible Ab–Ag complexes has occurred, for various reasons. If there are a large number of complexes formed ( 2), then the resolution window between free Ab and Ag may be too small to resolve these completely. Alternatively, if the complexes are rapidly interconverting with one another, i.e., the kinetics of formation and dissociation are rapid, perhaps too fast for the resolution time scale, then these species would always produce broad, unresolved peaks that are never really separable.

Figure 2 Electropherograms of IgG, hGH, and mixtures containing an excess of IgG. Experimental conditions used a fused silica capillary 100 cm long (80 cm to the detector) with 50 m i.d. and 360 cm o.d. The buffer was 0.1 M tricine, pH 8.0, applied voltage was 30 kV, injection volumes were 9 nl, detection was at 200 nm (52). (Reproduced with permission from the copyright holder, Elsevier Science Publishers and Journal of Chromatography.)

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We have used bovine serum albumin (BSA) as an Ag and its monoclonal, anti-BSA, Ab to perform a separation study of the complexes by CZE(20,59). In this example (Fig. 3), an AccuPure Z1 reagent was used to suppress any unwanted protein and uncoated, silica wall interactions. In the four sequences illustrated, the first two (a, b) illustrate injection of purified (affinity chromatography) anti-BSA (a) and then BSA (b), whereas panel (c) illustrates a mixture of Ag and Ab with anti-BSA Ab in excess. Peaks 1 and 2, it is assumed, represent the two possible Ag–Ab complexes formed in solution. We are assuming that the earlier eluting complex is due to a 1:1 complex and peak 2 is perhaps due to a 1:2 complex of Ab–Ag. In Fig. 3, panel (d), an excess of BSA was present, leading to residual BSA peak, and now what appears to be only a single Ab–Ag complex, probably the 1:2 (peak 2), having two BSA molecules complexed with each Ab species because there is no residual Ab present. The BSA–anti-BSA system is perhaps an ideal example to study by CZE methods. These complexes appear to be quite stable under the migration conditions and times. There are fairly narrow, well-resolved, complex peaks, suggesting a lack of interconversion on this time scale.

However, in the absence of true identification of these complexes, it is not 100% possible to identify each of the complex peaks (20,59). Indeed, in none of the existing CE immunoaffinity studies reported have any Ab–Ag complexes been identified by light scattering or mass spectrometric methods (60a,b). This would require, for example, a size exclusion chromatographic (SEC) separation of a particular complex and then on-line characterization with isolation and reinjection under CZE conditions (60c).

In the case of BSA–anti-BSA, the two complexes were well separated. These separation conditions may not, however, differentiate any microheterogeneity, if present, of the Ab. Alternatively, the microheterogeneity of the Ab may be so little that it cannot be distinguished by these CZE conditions.

In Shimura and Karger’s work (61) that dealt with the immunoassay (ACE or APCE) of hGH, they used CIEF to separate the complexes from Ag and Ab. To avoid any microheterogeneity of the Ab that might complicate their immunoassay, a Fab′ fragment from the Ab was used to interact with the Ag. The complexes thus formed and the excess tagged Fab′ fragments were all separated by CIEF. Figure 4 illustrates a set of typical CIEF electropherograms for this system, with conditions indicated. In panel (a), using a buffer without the Ag, only the two FL-tagged Fab′ species are present. In panel (b), now with met-rhGH first complexed with the TR-Fab′ species, two separate Fab′–Ag complexes appear, with perhaps some residual TR-Fab′ species/ peaks still present. The presence of two TR-Fab′ species, panel (a), illustrates a generic problem in using immunoaffinity (Ab) recognition in all of CE:

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Figure 3 A series of CZE electropherograms for bovine serum albumin (BSA), antiBSA, and complexes of BSA–anti-BSA. Operating conditions: uncoated capillary, 50m 70 cm, 40 cm to detector, 15 kV applied voltage, UV detection at 214 nm, vacuum injection 30 kpa-s, buffer 60 mM phosphate, pH 7.8, 1 M AccuPure Z1-methyl reagent (Waters), sample dissolved in phosphate buffer, pH 7.0. (a) Injection of antiBSA purified on protein G column first. (b) Injection of BSA, monomer purified by SEC first. (c) Mixture of BSA monomer and anti-BSA, with anti-BSA in excess. (d) Mixture of BSA monomer and anti-BSA, with BSA in excess (peak assignments: peak 1 1:1 complex; peak 2 1:2 complex, other peaks as indicated) (59).

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Figure 4 Example of an approach to immunoaffinity recognition in CZE using an FL probe Fab fragment of the Ab (61). CIEF analysis of crude TR-Fab′ and its complexes with met-rhGH. APCE was done with Pharmalyte 3–10 and using TR-Fab′ before purification as an affinity probe with (a) TTA-BSA buffer and (b) met-rhGH (1 µ g/ml) as samples. The free TR-Fab′ and the complexes are marked by * and **, respectively. (Reproduced with permission from the copyright holder, American Chemical Society and Analytical Chemistry.)