Development and Comparative Evaluation of Endolysosomal Proximity Labeling-based Proteomic Methods in Human iPSC-derived Neurons

ABSTRACT: Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic interaction networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, the challenge of selecting proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network, by knocking in (KI) an engineered ascorbate peroxidase (APEX2) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We systematically investigated and optimized the key parameters involved in proximity labeling MS to address the major challenges in the field. We also conducted comparative evaluation between this KI-LAMP1-APEX2 method and our overexpressed LAMP1-APEX2 probes, achieving complementary identification of both known and novel lysosomal membrane and membrane-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and interactions in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling technologies.

INSTRUMENT(S): Q-Exactive HFX

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ling Hao  

PROVIDER: MSV000086260 | MassIVE | Sat Oct 10 07:08:00 BST 2020

REPOSITORIES: MassIVE