Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSC-Derived Neurons
journal contributionposted on 17.11.2020, 21:04 authored by Ashley M. Frankenfield, Michael S. Fernandopulle, Saadia Hasan, Michael E. Ward, Ling Hao
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 protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the 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 (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batches of APEX labeling and different APEX probes. We conducted a comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.
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lysosomal functionsPCCAlysosomal-interacting proteinsendolysosomal networkproteomic experimentsoverexpression LAMP 1-APEX probesEndolysosomal Proximity Labeling-Ba...APEX probesKI-LAMP 1-APEX methodprotein networksendogenously biotinylated proteinsproteomics dataMStechniques offerHuman iPSC-Derived Neurons Proximit...proteomic microenvironmentiPSC-derived neuronslysosomal membraneendogenously biotinylated proteinmass spectrometryComparative Evaluationlysosome-associated membrane protein 1nanometer resolutionascorbate peroxidase