PROJECT SUMMARY
Newborn screening (NBS) is one of the most successful public health programs that provides cost-effective early
diagnosis of treatable disorders. The basis of NBS is to quantify a biomarker that is indicative of disease in a
dried blood spot (DBS). It was projected that 60 cell and gene therapies could be approved by the FDA within
10 years, which underscores an urgent need to modernize the NBS system. A major challenge for NBS of many
treatable genetic disorders is the unavailability of a biomarker in DBS, because there is no accumulation of small
molecules in patients. In this proposed studies, it is also not feasible to measure the biological function of the
protein (i.e. enzyme assay or other functional assays). Instead, reduction in the abundance of a specific protein
(encoded by the disease-causing gene) can be used for the diagnosis of genetic diseases as many genetic
conditions are caused by mutations that lead to non-functional proteins that are quickly degraded, as exemplified
in Wilson disease. Given the complexity of the DBS sample and the typical low-abundance nature of target
proteins, a technique called Immuno-SRM (Immunocapture coupled to selected-reaction-monitoring, or SRM)
had been successfully developed to quantify proteins in DBS. One drawback of our current Immuno-SRM for
NBS is that the LC-MS/MS time is about 3-4 min per sample. In this study, we propose a novel idea of expanded
multiplexing by using newborn-specific peptide mass tags to greatly increase the throughput of proteomic-based
NBS. Tagging of peptides with mass-differentiated tags allows samples from multiple newborns to be analyzed
in the same LC-MS/MS run. The objective of this application is to develop Immuno-SRM multiplex assay to
quantify 19 peptides for NBS of 12 genetic conditions including tuberous sclerosis. Successful implementation
of the project will enable the screening of 12 diseases simultaneously using a single DBS sample with a MS
instrument time < 1 min per sample. The goal will be achieved through the following specific aims. (1) Develop
an Immuno-SRM method to quantify TSC2 for tuberous sclerosis that is suitable for newborn screening.
Considering that we have already developed monoclonal antibodies (mAb, which is used to enrich surrogate
peptides for disease-specific proteins) for all conditions except TSC, the first aim is to raise mAb against TSC2
surrogate peptides and develop an Immuno-SRM for its quantification followed by optimization for multiplex
analysis. (2) Development of peptide tagging for increasing the throughput of Immuno-SRM assays.
Tagging of peptides with mass-differentiated tags allows samples from multiple newborns (>4 samples) to be
analyzed in the same LC-MS/MS run (equivalent to individual barcode sequences for next-generation-
sequencing). (3) Performance evaluation of the multiplexed Immuno-SRM method. First, the workflow will
be tested on known patient samples for its sensitivity. Second, a mini-pilot study on 3000 random newborn DBS
will be performed to test the robustness of the method.
