The NIMH-NINDS Clinical Proteomics Unit (CPU) is an element of the NINDS Translational Neuroscience Center (TNC). Our mission is twofold; first, we provide clinical investigators with access to state-of-the-art mass spectrometry instrumentation and bioinformatics tools required to incorporate proteomics into their research. Second, we invest a significant portion of our efforts in incorporating new sample preparation methods into our collaborative projects. Almost all of the collaborative projects engaged by CPU utilize immunoaffinity techniques (IP and Co-IP) to generate samples, e.g., from CSF, cell culture, or tissue lysate, for proteomic analysis. In the past year, we have analyzed IPs that have membrane proteins as the bait protein and hence require detergents to maintain solubility. CPU has adapted our on-bead digestion (OBD) protocol to allow inclusion of anionic detergents that can be completely removed by acidification and phase transfer into an immiscible organic solvent (upper phase), while digestion products remain in an aqueous phase (lower phase). After removal of the organic solvent, the aqueous phase is directly compatible with desalting by sold phase extraction. We anticipate broad utility of this methodology as several active projects are focused on integral and/or membrane-associated proteins and their interacting partners. Several collaborative projects have been recently published (see Bibliography) or are being prepared for submission. As an example, Dr. Avi Nath and colleagues in the Section of Infections of the Nervous System (SINS), have an active interest in understanding the physiological role of human endogenous retroviruses (HERVs), which are viral elements that were integrated into the human genome millions of years ago. SINS has demonstrated expression of HERV-K sequences in patients with Amyotrophic Lateral Sclerosis, and separately, neurotoxicity associated with HERV-K expression in differentiated neurons. Dr. David Wang and colleagues in the Neurodifferentiation Unit (TNC) characterized the expression and function of HERV-K env in IPS cells and in neuronal differentiation. IPs using HERV-K env as the bait protein were prepared from IPS cell lysate to identify protein interactions (manuscript in preparation). CPU advised Drs. Youle and Ward that their interest in quantitative proteomics projects necessitated the recruitment of a Ph.D. level scientist (IRTA Fellow) to focus specifically on these demanding projects. Dr. Ling Hao recently joined NINDS and is utilizing CPU instrumentation to specifically address these projects.
|Johnson, Tory P; Tyagi, Richa; Lee, Paul R et al. (2017) Nodding syndrome may be an autoimmune reaction to the parasitic worm Onchocerca volvulus. Sci Transl Med 9:|
|Kar, Amar N; Vargas, Jose Norberto S; Chen, Cai-Yun et al. (2017) Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking. Mol Cell Neurosci 80:32-43|
|Vargas, Jose Norberto S; Kar, Amar N; Kowalak, Jeffrey A et al. (2016) Axonal localization and mitochondrial association of precursor microRNA 338. Cell Mol Life Sci 73:4327-4340|