Enzymes probe-labeled

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Similar techniques can be used to devise avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawanefrz/., 1990 Lloyd etal., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

In this platform, antibodies that specifically recognize enzyme targets of ABPP are arrayed on glass slides and used as capture reagents. The application of probe-labeled proteomes on these slides results in the localization of fluorescence, which can be directly detected by fluorescence scanning [50]. The end result for this experiment is the consolidation of the isolation, detection, and identification of probe-labeled enzymes into one step (Fig. 6). 

In a second line of research, they prepared a new set of synthetic (3-lactam probes 62 around the monocyclic aztreonam structure (Fig. 28). Interestingly, none of these modified (3-lactam probes labeled any PBPs but had preference for other enzymes such as (3-ketoacyl acyl carrier protein III (KAS III), a (3-lactamase,... 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

Figure 7 (a) Capillary electrophoresis (CE) for ABPP. The probe-labeled proteome is trypsinized and peptide fragments are analyzed via CE. (b) General strategy for antibody-based ABPP microarrays. Proteomes are labeled in solution with fluorescent activity-based probes and captured on glass slides arrayed with enzyme-specific antibodies. Reproduced by permission of The Royal Society of Chemistry. 

To monitor protease activities in plant extracts, Wang etal.li8 generated biotinylated peptides that contained a /3-lactone reactive group. The probes labeled several enzymes in leaf proteomes of Arabidopsis thaliana. Interestingly, these studies led to the identification of a papain-like protease called RD21 that has the unexpected ability to ligate donor molecules such as peptides or lactones, probably through a thioester intermediate, to unmodified N termini of acceptor molecules. 

Figure 29 Competitive ABPP. Inhibitors are screened in whole proteomes and potent compounds are identified by their ability to reduce probe labeling of the target enzyme.

Many enzymes use the flavin cofactor at the active site the fluorescent active site approach can be applied to study these enzymes at the single-molecule level. Other naturally fluorescent enzymes, like those that contain NAD cofactors, can in principle be studied, although the bluer fluorescence of NAD poses a technical challenge for singlemolecule fluorescence detection. As the approach uses the natural fluorescence of the enzyme, no labeling with fluorescent probes is needed, offering no or minimum perturbation on the enzyme structure and function. 

---