The overall goal of this project is develop tandem mass spectrometry for newborn screening of the subset of lysosomal storage diseases for which treatment options exist or are being developed. A punch from a dried blood spot on a newborn screening card is used as a source of lysosomal enzymes, and this is incubated with a collection of substrates in a suitable buffer to allow formation of products. A second punch is used for extraction with organic solvent to recover biomarkers for lysosomal storage diseases. Both assays can be combined into a single multiplex tandem mass spectrometry assay for newborn screening of multiple treatable lysosomal storage diseases. We propose to pilot this highly multiplexed assay in the Washington State Newborn Screening Laboratory. We will also carry out exome DNA sequencing on dried blood spots that give an enzyme activity below the cut-off value. This data will allow us to determine the positive predictive values and false positive rates for the screening assay. These pilot studies will explore the feasibility of newborn screening of treatable lysosomal storage diseases. The second component of the project is to develop tandem mass spectrometry assays to support post- newborn screening analysis for lysosomal storage diseases. We will use high resolution assays to measure small changes in enzyme levels in blood-derived leukocytes so that the severity of the lysosomal storage disease can be estimated.