The NIMH-NINDS Clinical Proteomics Unit (CPU) was established on June 1, 2013, as part of the NINDS Translational Neuroscience Center. Our mission is to provide clinical investigators with access to state-of-the-art mass spectrometry instrumentation and evolving proteomics technologies. We are pursuing proteomic analysis of cerebrospinal fluid (CSF) as a source of potential biomarkers of neurological diseases. CPU maintains an active collaboration with Dr. Avindra Nath and his colleagues in the Section of Infections of the Nervous System, NINDS. For example, Dr. Tory Johnson sought to determine if an autoimmune process was contributing to the pathogenesis of Nodding Syndrome (NS), a distinctive epileptic disorder associated with brain atrophy of unknown etiology that occurs in epidemic form among children in East Africa. NS has a consistent epidemiological association with Onchocerca volvulus, the etiologic agent of onchocerciasis. Dr. Johnson found patients with NS had a 30,000 fold increase in antibodies to leiomodin-1 as compared to age- matched unaffected controls. Dr. Richa Tyagi demonstrated that autoantibodies to leiomodin-1 in the CSF of patients with NS were cross-reactive to O. volvulus. Screening of lysates prepared from adult male and female O. volvulus with pooled sera from patients with NS and control donors by Western blot analysis showed limited but differential immunoreactivity profiles. Bands to which patients with NS demonstrated increased immune reactivity were excised from the gel and identified by mass spectrometry. O. volvulus tropomyosin was one of the proteins identified. Tropomysosin proteins are known to play a role in neuronal structure and morphology, including dendritic and axonal branching and neurite outgrowth. Antibodies to leiomodin-1 from patients with NS cross react with O. volvulus tropomyosin and therefore suggest that O. volvulus infection and subsequent antibody production may result in the generation of leiomodin-1 reactive autoantibodies, establishing a putative mechanistic link between NS and O. volvulus. This work has been submitted for publication in The Lancet. In previous studies, Dr. Barry Kaplan and colleagues in the Molecular Neurobiology Section, NIMH, identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3-untranslated region of the cytochrome c oxidase subunit (COXIV) mRNA that was necessary and sufficient for the axonal localization of COXIV mRNA in superior cervical ganglion (SCG) neurons. Little is known about the proteins that regulate the axonal trafficking and local translation of the COXIV transcript. To identify proteins involved in the axonal transport of the COXIV mRNA, Dr. Amar Kar used this 38-nucleotide COXIV RNA zip-code as bait for RNA protein binding studies. Gel-shift assays of the biotinylated COXIV-zipcode incubated with retinoic-acid differentiated SHSY5Y cell lysates showed that the zipcode binds endogenous proteins and forms ribonucleoprotein complexes. CPU performed a mass spectrometric analysis of proteins isolated by affinity purification using biotinlylated COXIV-zipcode oligomers. These studies lead to the identification of a number of RNA-binding and mitochondria-associated proteins such as fused in sarcoma/translated in liposarcoma (FUS/TLS) and Parkinson disease protein 7 (PARK7/DJ-1) respectively. FUS/TLS and PARK7/DJ-1 are genes that have been previously linked to inherited cases of neurodegenerative disorders such as Amyotrophic lateral sclerosis and Parkinsons disease respectively. Validation using western blotting analyses confirmed the presence of the candidate proteins in the COXIV-zipcode affinity purified complexes from SHSY5Y lysates. In addition, using immunohistochemical and western blotting analyses, we also established the presence of these candidate COXIV-zipcode binding proteins in SCG neurons. This work has been submitted for publication in Molecular and Cellular Proteomics. Using this RNA affinity pulldown approach with mass spectrometric analyssis, Dr. Kaplan and colleagues have started to define the ribonucleoprotein complexes that regulate the trafficking and local expression of the COXIV mRNA in the axon. Future experiments are aimed at confirming the interaction of these candidate binding proteins with the endogenous COXIV mRNA in SCG neurons and assessing the in vivo significance of the candidate proteins in axonal transport and local translation of COXIV mRNA. Improvement in resolution, sensitivity and duty cycle in modern mass spectrometers continues to advance at an impressive pace and leads to revision of standard sample preparation methods. In particular, the on-bead digestion protocol developed in this lab was developed using a first generation Orbitrap instrument (low femtomole limit of detection) has been revised to a dual enzyme one pot sample preparation prior to two dimensional liquid chromatography (2D LC-MS/MS, high pH reversed phase HPLC off-line + nanoflow HPLC directly coupled to the mass spectrometer). Using an Orbitrap Elite instrument (low attomole limit of detection), we routinely identify a 300% increase in protein IDs albeit with a similar increase in instrument time. NINDS has addressed this decrease in sample throughput by adding two additional LC-MS/MS systems for FY2016.