Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSCDerived Neurons

作者：Ashley M. Frankenfield · 年份：2022 · DOI：10.1021/acs.analchem.0c03107.s002 · 被引用次数：1

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 Corresponding Author: Phone: +1 (202) 994-4492; linghao@gwu.edu. The authors declare no competing financial interest. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.0c03107. Detailed methods to develop LAMP1-APEX iPSC lines; additional figures for the fluorescence imaging of other APEX lines in neurons, optimization of protease amount, PCCA normalization, evaluation of false discoveries in KuB line, bead titration assay for other APEX lines, Venn diagram of all APEX proteomics and controls (PDF) Additional tables of the interference peptide exclusion list, whole cell lysate protein list for H2O2 vs PB vs Ctrl groups, protein IDs from all APEX proteomics datasets, and known lysosomal protein coverage in three LAMP1-APEX probes (XLSX) Comp...