The NIMH-NINDS Clinical Proteomics Unit (CPU) is an element of the NINDS Translational Neuroscience Center. 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 methods development. Specifically, we are developing a sample preparation methodology using cerebrospinal fluid (CSF) as a source of potential biomarkers of neurological diseases. Analogous to plasma, the concentration of protein constituents in CSF spans 8-10 orders of magnitude and >90% of the total protein mass is composed of 12-20 highly abundant protein species. The dynamic range of state-of-the-art mass spectrometers is 10E2-10E3 in full scan mode and as high as 10E4-10E5 in selected ion monitoring mode. The analytical problem is how to produce a census of protein constituents in a sample that is significantly more complex than current instrumentation can handle. Our proposed solution utilizes an automated platform for depletion of highly abundant proteins (albumin and IgG), digestion of the remaining proteins using an immobilized enzyme reactor, and capture of the digestion products (peptides). Individual samples are then derivatized with an isobaric mass tag, which allows multiplexing of up to ten individual samples prior to multi-dimensional LC-MS/MS. We have made significant progress in the last year. Specifically, we have optimized conditions for affinity depletion of both albumin and IgGs to >95%. Our database of proteins identified in CSF samples has 4,100 entries, which is approximately double the number of entries from FY2015 and reflects improvements in our methodology and use of an Orbitrap Fusion Lumos mass spectrometer that was acquired with support from the Office of AIDS Research. We are currently assessing run-to-run reproducibility prior to conducting pilot experiments using clinical samples, specifically age/gender matched control vs HIV CSF samples from the NINDS CSF Bank. Almost all of the collaborative projects the CPU has undertaken utilize immunoaffinity techniques (IP and Co-IP). The on-bead digestion (OBD) protocol developed in this lab was based on the idea that proteins isolated by IP/Co-IP would coated in layers, the outermost layer being loosely bound and the innermost layer being tightly associated with covalently bound antibodies. The OBD protocol yields four fractions from each sample by using increasingly vigorous conditions. Fractions were analyzed by LC-MS/MS sequentially using a first generation hybrid Ion Trap-Orbitrap instrument and the data files were combined to yield a census report of 300 protein IDs for a given sample. We have revised the OBD protocol to exploit the gain of 2-3 orders of magnitude in sensitivity using current generation state-of-the-art instrumentation. The revised OBD protocol is a dual enzyme (LysC and trypsin), one pot sample preparation prior to 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 (900 IDs) albeit with a commensurate increase in time.

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