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 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 batch of APEX labeling and different APEX probes. We conducted 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 iPSCderived 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.

A Cellular Taxonomy of the Adult Human Spinal Cord

“…We have previously described the enrichment of biotinylated proteins and bead titration assay 91 , Briefly, total protein concentration of spinal cord lysates was measured by the DC Protein Assay (Bio-Rad Cat # 5000111). We aliquoted 1 mg proteins per replicate and incubated them with 250 µL pre-washed streptavidin magnetic beads at 4 °C for 12-h to enrich biotinylated proteins.…”

Protein Contaminants Matter: Building Universal Protein Contaminant Libraries for DDA and DIA Proteomics

“…Streptavidin coated beads generated overwhelming streptavidin peptide signals which is consistent with our previous findings. 6 These exogenous contaminant proteins are often originated from a different species which will not be identified unless the contaminant FASTA library is included in the data analysis workflow.…”

Clinical Insights into Mitochondrial Neurodevelopmental and Neurodegenerative Disorders: Their Biosignatures from Mass Spectrometry-Based Metabolomics

“…Besides patient samples, cellular and animal models are often used to investigate the molecular mechanisms underlying mitochondrial diseases. Primary patient-derived cells and human cell lines are most accessible and quickly available for genome editing to model disease-relevant mutations in immortalized lymphoblastoid cells, cybrid cells, HeLa cells, HEK293 cells, stem cells, and stem cell-derived neurons [ 106 , 107 , 108 ]. Animal models, such as engineered mice, Drosophila, and Caenorhabditis elegans, provide in vivo platforms to study the underlying pathogenic mechanisms but fail to recapitulate the clinical symptoms of a specific syndrome [ 109 ].…”