DiDBiT-TMT:\nA Novel Method to Quantify Changes in\nthe Proteomic Landscape Induced by Neural Plasticity

Direct detection of biotinylated proteins (DiDBiT) is\na proteomic\nmethod that can enrich and detect newly synthesized proteins (NSPs)\nlabeled with bio-orthogonal amino acids with 20-fold improved detectability\ncompared to conventional methods. However, DiDBiT has currently been\nused to compare only two conditions per experiment. Here, we present\nDiDBiT-TMT, a method that can be used to quantify NSPs across many\nconditions and replicates in the same experiment by combining isobaric\ntandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain\nslices to determine changes in the de novo proteome that occur after\ninducing chemical long-term potentiation (cLTP) or treatment with\nthe neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT’s\ncapacity to quantitatively compare up to 9 samples in parallel. We\nshowed that there is a minimal overlap among NSPs that are differentially\nexpressed in cLTP-treated organotypic brain slices, norepinephrine-treated\norganotypic brain slices, and organotypic slices undergoing combinatorial\ntreatment with norepinephrine and cLTP. Our results point to the possible\ndivergence of the molecular mechanisms underlying these treatments\nand showcase the applicability of DiDBiT-TMT for studying neurobiology.