Multiplex Analysis of Inborn Errors of Metabolism

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DESCRIPTION (provided by applicant): Enzyme deficiencies are the major cause of genetic diseases. In spite of recent advances in nucleic acid screening technologies that elucidate correlations between genetic alterations, protein expression, and function, it is not yet practical to diagnose individual patients by sequencing their full-length genes. Enzyme analysis of tissue or blood cells remains the preferred standard for measurements of protein function to obtain confirmation of suspected disorders. The main focus of the current proposal is to develop several rapid, generally-useful, accurate, and sensitive enzyme assays based on electrospray ionization mass spectrometry as a single instrumental platform. The strategy is to quantify enzymatic reaction velocities in cultured cell lysates by observing mass changes resulting from an enzyme action on a synthetic substrate-conjugate. Biotin is used as a molecular handle in substrate-conjugates, that allows facile and selective separation of enzymatic products from complex biological mixtures by reversible capture with immobilized streptavidin or monomeric avidin, followed by release for mass spectrometric analysis. Quantitation is achieved by using biotinylated internal standards that are chemically identical to the products of enzymatic reactions, but are distinguished by different molecular mass due to the presence of stable heavy isotopes. Previous results for lysosomal storage diseases showed that this approach allows detection and quantitation of enzymatic products in as little as 2500 cells, makes it possible to analyze two or more enzymatic reactions in a single reaction mixture, and the analytical procedure is readily automated. The proposed work is aimed at developing assays for the enzymes phosphomannomutase, phosphomannoisomerase, dolichol-P mannose synthase, and GlcNAc transferase II to achieve specific diagnoses of the various forms of congenital deficiencies of glycosylation. Another complex group of disorders that will be targeted are the porphyrias that often present perplexing clinical symptoms. The results from the new assays and those developed previously for lysosomal storage diseases will be transferred from the research laboratory to clinical practice. A new technology for the detection of low-level proteins, called Visible Isotope Coded Affinity Tags (VICAT), will be developed to quantify the levels of the structural protein dystrophin, whose deficiency causes Duchenne and Becker muscular dystrophies. In addition to this disease-related application, the VICAT technology will be applicable as a general method for sensitive, specific, and absolute quantitation of cellular proteins.