The majority of recognized genetic diseases are caused by enzyme deficiencies. The advent of molecular biology has allowed a more detailed explanation of how genetic alterations influence protein function, but in most cases DNA analysis will remain a second tier approach to the clinical confirmation of suspected disorders. Enzyme analysis of an appropriate tissue will remain the preferred standard as a measurement of protein function for diagnosis. There are numerous assays of enzyme functions in use, many are tedious, and a variety of different analytical techniques are used to assay the catalytic reactions. The focus of the current proposal is to develop a generally-useful, accurate, and sensitive enzyme assay based on electrospray mass spectrometry. The strategy is to quantify enzymatic reaction velocities by observing the change in mass that the substrate undergoes during the enzymatic reaction. Substrates conjugated to a molecular handle, such as biotin, will be used so that the enzymatic product can be easily purified for mass spectrometry from complex biological fluids by capture with a handle-specific receptor such as streptavidin. With this method, it will be possible to analyze several enzymatic reactions in a single reaction mixture using a single injection into a mass spectrometer (multiple analysis). Electrospray ionization mass spectrometry is an extremely sensitive analytical technique (sub-picomole detection is routinely achieved), and can thus be applied in the situation where only small amounts of biological sample are available. This novel approach has been successfully tested by quantitatively assaying beta-galactosidase in cells from a healthy patient and from an individual lacking this enzyme. The proposed work will be directed at developing novel analyses for the four enzymes that define the Sanfilippo syndrome, two lipid hydrolyzing enzymes that define Niemann-Pick (types A and B) and Krabbe diseases, and two enzymes in the tyrosine breakdown pathway that define tyrosinemia. The development of enzyme assays for several diverse enzymes will allow for multiple enzyme analyses from a single reaction to correctly identify a deficient enzyme that is responsible for similar-appearing genetic disorders and with sensitivity that equals or exceeds those of current methods.
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