UC San Diego

The Cline Lab has used N-hydroxysuccinimidobiotin (NHS-Biotin) in proteomic analysis of the visual system by labeling neuronal projections from the eye to the Superior Colliculus (SC) and the Lateral Geniculate Nucleus (LGN), in adult rats in vivo. Using intravitreal injections, NHS-Biotin labels endogenous proteins in the retina that are then transported to various retinal targets in the visual system. NHS-Biotin protein labeling allows the identification and investigation of the population of proteins transported in long-range axonal projections. The covalently bound biotin is used as a tool for immunoprecipitation to conduct biochemical and proteomic analyses.  Here, we are interested in adapting the NHS-Biotin protein labeling technique to allow precise study of transportomes in the developing mammalian nervous system in vivo. We tested whether NHS-Biotin can be used to study projections in the developing brain, tracing connectivity, as well analyzing the proteomic composition by mass spectrometry. We addressed these questions in the following experiments by conducting unilateral intracortical injections of NHS-Biotin into post-natal day 3 rat pups.  We used western blots to compare the levels biotinylated protein in the injected (ipsilateral) and contralateral cortical hemispheres.  We probed NeutrAvidin enriched biotinylated endogenous proteins for known axonal proteins, such as MAPK, Tau, and β-synuclein, and found that they showed significant intercortical transport via the corpus callosum, anterior commissure and cortical-spinal tract. These data demonstrated that NHS-Biotin can be used in the morphological and biochemical study of long-range axonal projections in the developing rodent brain following targeted injections. To test whether we could use the NHS-Biotin labeling strategy for mass spectrometric proteomic analysis of the developing cortical transportome, we used the Direct Detection of Biotin-containing Tags (DiDBiT), which increases the sensitivity of biotinylated protein detection. The mass spectrometry data show the proteomic landscape of the axonally transported proteins across the corpus callosum.  This paper reviews previous labeling strategies and limitations of these techniques, while introducing a novel strategy that overcomes many of these limitations. The experiments we conducted highlight how the NHS-Biotin labeling strategy allows efficient identification and investigation of axonally transported proteins.