Analysis of Rates of Multiple Enzymes in Cell Lysates by Electrospray Ionization Mass Spectrometry

Deficiency of enzymatic activities is a major cause of genetic defects1 as documented by maladies such as phenylketonuria,2 galactosemia,3 or glycogen storage disease.4 Monitoring enzyme functions by biochemical assays is an essential diagnostic tool that employs a multitude of analytical techniques including spectrophotometric, fluorometric, and radiometric detection of products. Although these methods are quite sensitive, they do not provide chemical speciation of products and therefore are difficult to use for assaying several enzymes simultaneously in a single sample. Mass spectrometry for quantification of a collection of metabolites in biological fluids has emerged as a powerful approach for the analysis of birth defects,5 but this analytical technique has not been developed for the direct analysis of rates of individual enzymatic steps. Here we describe a new analytical method for monitoring and quantification of enzymatic activities in cell homogenates that has the sensitivity required for biomedical applications, permits simultaneous (multiplex) monitoring of multiple reactions, and can be readily automated. A key feature of the new method is the use of electrospray ionization mass spectrometry (ESI-MS)6 for the simultaneous detection of enzymatic products and chemically identical internal standards, which are distinguished by stable isotope (deuterium) labeling. The second feature is the use of substrate conjugates combined with affinity purification for facile capture of enzymatic products from crude biological fluids. The method starts with the design of a synthetic conjugate molecule that contains a target substrate for the desired enzyme that is covalently attached to a linker, which in turn is attached to a molecular handle. Action of enzyme on the substrate conjugate causes cleavage or other modification that changes its molecular mass (Scheme 1). The change of mass is detected by ESI-MS. The linker and handle are designed to serve several key functions, e.g., to facilitate ionization by ESI, to block action of other enzymes in the biological fluid, and to allow highly selective capture from the complex matrix for facile purification. An example of this approach is the design and synthesis of conjugates 1 and 2 (Scheme 2) to simultaneously assay lysosomal â-galactosidase and N-acetyl-R-D-glucosaminidase, respectively. Deficiency of the former enzyme results in one of the lysosomal storage diseases, GM1-gangliosidosis, a condition that occurs in the population with a frequency of about 1 in 50 000 and leads to early death of affected children.7 Deficiency of N-acetyl-R-Dglucosaminidase results in the rare lysosomal storage disorder Sanfilippo syndrome type B.8 Conjugates 1 and 2 consist of biotin as a molecular handle, which is coupled to sarcosine. Biotin allows highly specific capture of the substrate conjugate through noncovalent binding to streptavidin immobilized on agarose beads.9 Sarcosine provides an N-methylated amide linkage to biotin to block the enzyme biotinidase, which is often present in the cellular fluids and could cause cleavage of the conjugate molecule during the assay.10 In addition, we found that biotinyl-sarcosine conjugates can be displaced from streptavidin by addition of biotin. The N-biotinylsarcosine block is linked to a polyether diamine, the length of which can be varied to avoid mass/charge overlaps of products and internal standards. The linker also allows facile introduction of multiple deuterium atoms (i.e., 8 deuteriums in 5 and 4 in 6, Scheme 2)11 to permit the synthesis of internal † Department of Chemistry. ‡ Department of Pediatrics. § Department of Biochemistry. * To whom correspondence should be addressed. (1) Scriver, C. R.; Beaudet, A. L.; Sly, W. S.; Valle, D. The Metabolic and Molecular Bases of Inherited Disease; McGraw-Hill: New York, 1995. (2) Scriver, C. R.; Kaufman, S.; Eisensmith, R. C.; Woo, S. L. C. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 10151076. (3) Segal, S.; Berry, G. T. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 967-1000. (4) Chen, Y.-T.; Burchell, A. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 935-966. (5) For example, see: Morris, A. A. M.; Turnbull, D. M. Curr. Opin. Neurol. 1994, 7, 535-541. (6) Cole, R. B. Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Practice; Wiley: New York, 1997. (7) Okada, S.; O’Brien, J. S. Science 1968, 160, 10002. (8) Neufeld, E.; Muenzer, J. The mucopolysaccharidoses. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 2465-2494. (9) Bayer, E.; Wilchek, M. Methods Enzymol. 1990, 184, 49-51. (10) Wilbur, D. S.; Hamlin, D. K.; Pathare, P. M.; Weerawarna, S. A. Bioconjugate Chem. 1997, 8, 572-584. (11) The d8-linker was made by reacting DOCH2CH2OCH2CH2OD with CD2dCDCN in benzene with catalytic NaOD (Ashikaga, K.; Ito, S.; Yamamoto, M.; Nishijima, Y. Bull. Chem. Soc. Jpn. 1988, 61, 2443-2450), and the resulting dinitrile was reduced to the diamine with Ra-Ni. The d4linker was made in the same way using ethylene glycol and CD2dCDCN in CH3CN and catalytic NaOH. Scheme 1