Capturing Lysosomal Dynamics with Multifaceted Mass Spectrometric Strategies

Description:
Lysosomes are acidic vacuoles that digest and recycle a variety of intracellular and exogenous cargo. Lysosomes are highly dynamic and frequently interact with other cellular components in a transient fashion, which are difficult to capture with traditional immunoprecipitation and organelle isolation methods. Here, we developed a set of mass spectrometry (MS)-based proteomic and metabolomic strategies to characterize the dynamic lysosomal biology and evaluate lysosomal dysfunctions related to neurodegenerative diseases both in vitro and in vivo.

To capture the dynamic lysosomal interaction with other cellular components, we developed an endogenous lysosomal proximity labeling method targeting on the bait protein, lysosomal-associated membrane protein1 (LAMP1) in human pluripotent stem cells (iPSCs)-derived neurons. We also extended this method to fixed mouse brain tissues and identified lysosomal membrane proteins and lysosomal interacting proteins both in vitro and in vivo. We conducted a comprehensive investigation and optimization of the key parameters in proximity labeling workflow to solve key challenges in the field, greatly improving the accuracy, specificity, and reproducibility of proximity labeling MS. To characterize lysosomal content, we isolated intact lysosomes from iPSC-neurons to profile the proteins and small molecules inside lysosomes via MS-based proteomics and metabolomics strategies. To evaluate the global protein turnover, we implemented dynamic SILAC methods to measure protein half-lives in human neurons and the perturbations due to lysosomal dysfunction.

Speaker: Ling Hao - George Washington University
Dr. Ling Hao is an Assistant Professor in the Chemistry Department at the George Washington University in Washington, D.C. She received her PhD from the University of Wisconsin-Madison followed by a postdoctoral fellowship at the National Institutes of Health (NIH) before joining GW in 2019. Research in the Hao Lab is focused on developing MS-based proteomics, metabolomics, and proximity labeling techniques to understand lysosomal and mitochondrial dysfunctions underlying brain diseases. She has been recognized as a Ralph E Powe Junior Faculty Enhancement Award from Oak Ridge Associated Universities in 2020, an Emerging Investigator by the Journal of American Society for Mass Spectrometry (JASMS) in 2021 and a Rising Star Award by the Human Proteome Organization (HUPO) world congress in 2022.