General Protease Assay Method Coupling Solid-Phase Substrate Extraction and Capillary Electrophoresis Douglas B. Craig, ² Jerome C. Y. Wong, Robert Polakowski, and Norman J. Dovichi* Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incu- bated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unre- acted substrate to dramatically lower the background signal. The product is detected by capillary electrophore- sis, which provides powerful discrimination against prod- ucts generated by nonspecific proteases. With chymot- rypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 × 10 -13 M) chymotrypsin, or 5 amol of enzyme in the 10-μL sample volume. Proteases are enzymes that catalyze the hydrolysis of amide bonds of proteins and peptides. Proteases are involved in many biological processes including, but not limited to, cancer biology, 1 immune response, 2 viral replication, 3 neurochemistry, 4 and apo- ptosis. 5 Increased interest in proteases motivates the development of better techniques for their assaying. There are several methods to assay proteases. 6 Immunoassays target the protein but do not measure the activity of the protease. Antibodies can be difficult to generate. Also, antibodies can cross- react with other molecules, reducing the specificity of the assay. Most assays measure the enzyme activity. In one case, fluorogenic protease substrates are constructed from a fluorescent probe and a quencher that are located on opposite sides of the cutting site. When the protease cuts the substrate, the probe is liberated, which generates a highly fluorescent product. 7-9 The method is sensitive to nonspecific proteases that cut the substrate at other sites between the quencher and probe. Several assays detect the liberated hydrolysis product. An enzymatic method has been used to detect amino acids liberated by a protease. 10 The hydrolysis product can also be detected by use of an immobilized peptide that has a radioactively labeled free- solution end; appearance of radioactivity in the supernatant is evidence for the presence of a peptide. 11 Similarly, a peptide can be engineered that is overall neutral in charge but contains a highly charged, radioactively labeled fragment that is liberated upon cleavage. The cleaved fragment is captured on an ion- exchange resin and detected through its radioactivity. 12 These methods are all sensitive to nonspecific proteases. As a last method, the cleaved peptide can be analyzed by SDS- PAGE. This method is quite selective but not very sensitive. 6 The object of our study was to develop a protease assay method that combines the state-of-the-art sensitivity produced by laser- induced fluorescence with the selectivity provided by capillary electrophoresis. The method is general enough that it can be easily adapted for use with virtually any protease. Capillary electrophoresis (CE) utilizing postcapillary laser-induced fluores- cence detection (LIF) in a sheath flow cuvette is capable of detecting single fluorescent molecules after electrophoretic sepa- ration. 13 CE-LIF has also been used for the assaying of the single enzyme molecules of both alkaline phosphatase 14 and -galactosi- dase. 15,16 ² Present address: Department of Chemistry, University of Winnipeg, Win- nipeg, MB, Canada. (1) Tsuda, T. T.; Kodama, A.; Yamamura, M.; Matsuzaki, S.; Tsuda, T. FEBS 1993, 319, 35-9. (2) Jenne, D. E.; Tschopp, J. Immunol. Rev. 1988, 103, 53-71. (3) Blundell, T. L.; Lapatto R.; Wilderspin, A. F.; Hemmings, A. M.; Hobart, P. M.; Danley, D. E.; Whittle, P. J. Trends Biochem. Sci. 1990, 15, 425-30. (4) Lee C. M. Ciba Found. Symp. 1982, 91, 165-85. (5) Nicholson, D. W.; Ali, A.; Thornberry, N. A.; Vaillancourt, J. P.; Ding, C. K.; Gallant, M.; Gareau, Y.; Griffin, P. R.; Labelle, M.; Lazebnik, Y. A.; Munday, N. A.; Raju, S. M.; Smulson, M. E.; Yamin, T.-T.; Yu, V. L.; Miller, D. K. Nature 1995, 376, 37-43. (6) Hellen, C. U. Methods Enzymol. 1994, 241, 46-58. (7) Florentin, D.; Sassi, A.; Roques, B. P. Anal. Biochem. 1994, 141, 62-9. (8) Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science 1990, 247, 954-8. (9) Meldal, M.; Svendsen, I.; Breddam, K.; Auzanneau, F. I. Proc. Natl. Acad. Sci. U.S.A. 1990, 91, 3314-8. (10) Cathers, B. E.; Schloss, J. V. Anal. Biochem. 1996, 241,1-4. (11) Wondrak, E. M.; Copeland, T. D.; Louis, J. M.; Oroszlan, S. Anal. Biochem. 1996, 188, 82-5. (12) Hyland, L. J.; Dayton, B. D.; Moore, M. L.; Shu, A. Y.; Heys, J. R.; Meek, T. D. Anal. Biochem. 1990, 188, 408-15. (13) Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1996, 68, 690-6. (14) Craig, D. B.; Arriaga, E.; Wong, J. C. Y.; Lu, H.; Dovichi N. J. J. Am. Chem. Soc. 1996, 118, 5245-53. (15) Craig, D.; Arriaga, E. A.; Banks, P.; Zhang, Y.; Renborg, A.; Palcic, M. M.; Dovichi, N. J. Anal. Biochem. 1995, 226, 147-53. (16) Craig, D.; Dovichi, N. J. Can. J. Chem. Submitted. Anal. Chem. 1998, 70, 3824-3827 3824 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998 S0003-2700(98)00106-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/20/1998