Chemical Probe-Enabled Lipid Droplet Proteomics

Emerging evidence indicates that lipid droplets (LDs) play important roles in lipid metabolism, energy homeostasis, and cell stress management. Notably, dysregulation of LDs is tightly linked to numerous diseases, including lipodystrophies, cancer, obesity, atherosclerosis, and others. The pivotal physiological roles of LDs have led to an exploration of research in recent years. The functions of LDs are inherently connected to the composition of their proteome. Current methods for profiling LD proteins mostly utilize LD fractionation, including those based on proximity-based labeling techniques. Global profiling of the LD proteome in live cells without the isolation of LDs is still challenging. Herein, we disclose two small-molecule chemical probes, termed <b>LDF</b> and <b>LDPL</b>. Both <b>LDF</b>/<b>LDPL</b> are small in size and could freely and specifically migrate within the lipid context of LDs. Consequently, they were successfully used for live-cell fluorescence imaging of LDs and from animal tissues. We further showed that <b>LDPL</b> was capable of large-scale profiling of the LD proteome without the need of LD isolation. By using <b>LDPL</b>, 1584 high-confidence proteins, most of which could be annotated to prominent LD functions, were next identified. Importantly, further validation studies by using representative "hit" proteins revealed that CHMP6 and PRDX4 could act as the lipophagy receptor and lipolysis suppressor, respectively. Our results thus confirmed for the first time that <b>LDPL</b> is a powerful chemical tool for in situ profiling of LD proteomes. With the ability to provide a deeper understanding of LD proteomics from the native cellular environments, our newly developed strategy may be used in future to decipher the dynamics and molecular mechanism of LDs in various diseases.