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        <Description>Only half of individuals with suspected rare diseases receive a definitive genetic diagnosis following genomic testing. A genetic diagnosis allows access to appropriate patient care and reduces the number of potentially unnecessary interventions and related healthcare costs. Here, we demonstrate that an untargeted quantitative mass-spectrometry approach quantifying &gt;6,000 proteins in primary fibroblasts representing &gt;80% of known mitochondrial disease genes can provide functional evidence for 88% of individuals in a cohort of known primary mitochondrial diseases. We profiled &gt;90 individuals, including 28 with confirmed disease and diagnosed 6 individuals with variants in both nuclear and mitochondrial genes. Lastly, we developed an ultra-rapid proteomics pipeline using minimally invasive peripheral blood mononuclear cells to support upgrade of variant pathogenicity in as little as 54 hours in critically ill infants with suspected mitochondrial disorders. This study supports the integration of a single untargeted proteomics test into routine diagnostic practice for the diagnosis of rare genetic disorders in clinically actionable timelines, offering a paradigm shift for the functional validation of genetic variants.</Description>
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