Astrocyte Secretome Profiling via Biorthogonal Labeling Unveils Novel Factors Relevant to Neurodegenerative Diseases

Secreted proteins are key mediators of intercellular communication in multicellular organisms. However, progress in secretomics has been hindered by the lack of effective methods for capturing secreted proteins. Here, we present a two-step secretome enrichment method (tsSEM) that integrates unnatural amino acid labeling with click chemistry-based biorthogonal reaction, enabling robust in vitro secretome profiling in the presence of serum. Applying tsSEM, we systematically analyzed the secretome of human induced pluripotent stem cells (iPSCs)-derived astrocytes (iAst) across various disease models and identified a panel of astrocyte-secreted proteins contributing to noncell autonomous neurotoxicity. Among these, we validated two novel neurotrophic factors, FAM3C and KITLG, which enhanced neurite outgrowth, protected neuronal viability, and promoted neural progenitor proliferation. Our findings demonstrate the utility of tsSEM for high-resolution secretome analysis and underscore the potential of iAst-derived secretomes in elucidating disease mechanisms and identifying therapeutic targets.