Mitochondrial diseases are one of the most common types of inherited metabolic disorders and affects approximately 1 in 5,000 live births. Most routine diagnoses for mitochondrial disease rely on exome (ES) or genome sequencing (GS) and typically achieve a yield of ~50% of cases. We have been applying quantitative proteomics to help achieve a molecular diagnosis for patients with suspected mitochondrial disease and other rare diseases. From a small cohort of N=10 long term undiagnosed patients, we achieved an 80% diagnostic rate with our proteomics pipeline. Beyond the diagnostic component of our research, we are also interested in further exploring the data acquired from thousands of proteins and from a diverse range of clinical cases to provide insights into new protein function and biological processes. For instance, we have analysed lymphoblasts from a patient harbouring pathogenic variants in the mitochondrial alanyl tRNA synthetase (AARS2). The patient presented with reduced abundance of Complex IV and mitoribosome proteins. Further analysis of the proteomics data led us to investigate the function of HIGD2A, a hypoxia inducible gene 1 domain family member. Using CRISPR/Cas9 we generated HIGD2A knockout HEK293T cells, which presented with an isolated mitochondrial Complex IV defect and impaired mitochondrial respiration. Characterisation of HIGD2A function using a new pulse-chase SILAC technique revealed that this protein is specifically involved in the assembly of the COX3 module of the mitochondrial Complex IV. HIGD2A is the first assembly factor characterised to be required for the assembly of the COX3 module of Complex IV, despite over 20 other assembly factors previously been identified to be involved in the assembly of Complex IV. While no patients have been identified with mutations in HIGD2A to date, our data support the inclusion of HIGD2A as a candidate gene in clinical investigation of patients with Complex IV enzyme defect. Hence, our studies are an example of the power of further investigating clinical data to guide the discovery of novel protein function.