Cell-type-specific Proteomics in Human Brains Using Immunopanning and Antibody-Based Proximity Labeling

In the realm of neuroscience, the quest for comprehending the intricacies of the brain at a cell-type- specific and single-cell resolution is steadfast. However, this pursuit is impeded by the challenges posed by achieving accurate tissue isolation and acquiring fresh brain samples. Thus far, cell-type-specific human brain proteomes remain elusive. This poses a substantial knowledge gap in understanding protein expression and RNA-protein correlation at the cell-type-specific and single-cell levels. Moreover, the absence of reference proteomes tailored to distinct cell types within the human brain accentuates this knowledge gap, as these reference proteomic data are essential for emerging fields like subcellular proteomics and single-cell proteomics. In this pilot project, I will establish two parallel isolation and proteome methods to study cell-type-specific proteomics in both fresh and postmortem human brain samples. Firstly, I will employ innovative immunopanning techniques to isolate different cell types in fresh human brains. Subsequently, cell-type specific DDA- and DIA-based LC-MS-MS proteomics will be applied to analyze these samples. To address potential protein loss during tissue isolation, I will utilize a novel antibody-based proximity labeling proteomics pipeline that I previously established to uncover proteins in cell-type-specific compartments, such as cell bodies and processes, in postmortem human brain sections. By comparing the proteome datasets obtained from immunopanning and proximity labeling in fresh and postmortem brain samples, I aim to gain insights into the cell-type-specific proteome in human brains. Ultimately, this work seeks to establish high-quality cell-type-specific reference proteomes in human brains. These reference datasets will be instrumental in understanding the molecular basis of human brains, illuminating RNA-protein correlations, and advancing data analysis in subcellular proteomics and single-cell proteomics in humans. Furthermore, the project will provide valuable tools and resources to enhance our understanding of human brains in both health and diseases, such as drug addiction.