MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v03/MSV000086260/primaryOK2000000ProteomicsLing HaoQ-Exactive HFXHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=b5efe5727b924529888b5207e1e4ae83linghao@gwu.eduThe George Washington UniversityMassIVE52cysteine carbamidomethylationbiotin-phenol modification of tyrosineoxidation of methionineacetylation of protein N-terminusProximity-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.Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSC-Derived Neurons.Frankenfield Ashley M AM, Fernandopulle Michael S MS, Hasan Saadia S, Ward Michael E ME, Hao Ling Lhuman being, Nerve Cells, Man (Taxonomy), Procedures, postnatal development, Modern, Nerve, postnatal growth, growth and development, labeling, Methodological, procedures, Procedure, Nerve., Methodological Study, human, Cell, Human, Study, development, Techniques, iPSC, Methodological Studies, Homo sapiens, Method, Modern Man, Cells, Studies, Neuron, techniques, iPS cell, Nerve Cell, growth, Technique, Man, humans, methodologyF24B9.2, lysosome membrane, Materials, human being, apexl2, Procedures, experimental, Peptidomics, False, MEE6, Addresses, Nerve, number, Gene, LAMP-1, protein, Organelle, Spectrum Analyses, protein-containing complex, Migrant Workers, dIKK-gamma, Human, Membrane Tissues, protein polypeptide chains, method, Techniques, ZGRF2, LAMPA, organ field, polypeptide chain, Homo sapiens, Method, integral component of membrane, method used in an experiment, DmIKK-gamma, Gene Products, Mass, Studies, field, Analysis, dmIKKgamma, iPS cell, protein aggregate, IKK[[gamma]], Technique, Man, IKKg, Mass Spectroscopy, KEY, Key, L Ascorbic Acid Peroxidase, Mass Spectrum Analysis, study, F, methods, L-Ascorbic, Migrant Worker, Genetic, Man (Taxonomy), Acid Peroxidase, Analyses, Tissues, experimental section, developmental field, Transients, membrane region, labeling., Nonmigrant, Tissue, ATAPX1, Nonmigrants, labeling, proteins, Worker, Peroxidase, Transient, Gaps, Squatter, Study, CD107a, LGP120, Methodological Studies, IKK, LGP-120, Squatters, F24B9_2, region of membrane, Neuron, Peroxidases, ascorbate peroxidase 1, Nerve Cell, APEXL2, C430040P13Rik, Membrane Tissue, membrane, Ascorbate, protein complex, Arts, Modern, Proteins, integral to membrane, future organ, Procedure, membranous organ component, nanometre, maternal effect embryo arrest 6, Cistrons, Spectrum Analysis, Cell, Spectroscopy, P2B, AI196048, IKKgamma, DmIKKgamma, MS, Lamp-1, iPSC, native protein, Prgs, natural protein, dIKK, Migrant, Protein, Kenny, Nomad, whole membrane, Genetic Materials, Workers, Lysosomal membrane glycoprotein A, Migrants and Transients, Mass Spectrum Analyses, L-Ascorbic Acid Peroxidase, Genetic Material, nm, CS1, Mass Spectrum, Industrial, transmembrane, membrane of organ, Nerve Cells, Industrial Arts, 120 kDa lysosomal membrane glycoprotein, Ascorbate Peroxidase, Dmikkgamma, CD107 antigen-like family member A, ASCORBATE PEROXIDASE, XTH2, IKK-gamma, Spectrometry, L-Ascorbic Acid, INSDC_feature:gene, Methodological, Membrane, CG16910, Methodological Study, Lysosome-associated membrane protein 1, human, experimental procedures, Protein Gene Products, plan specification, Gene Proteins, APE2, DmelCG16910, Material, Nomads, Modern Man, cardinality, Cells, ape2, ATAPX01, Migrants, Cistron, LGP-A, humans, xth2human being, Nerve Cells, Man (Taxonomy), Procedures, postnatal development, Modern, Nerve, postnatal growth, growth and development, labeling, Methodological, procedures, Procedure, Nerve., Methodological Study, human, Cell, Human, Study, development, Techniques, iPSC, Methodological Studies, Homo sapiens, Method, Modern Man, Cells, Studies, Neuron, techniques, iPS cell, Nerve Cell, growth, Technique, Man, humans, methodologyF24B9.2, Materials, Product, APX, MEE6, LAMP-1, protein, Organelle, sci, Techniques, protein polypeptide chains, Method, Biological, HOW, How, Analysis, Biological Product, iPS cell, protein aggregate, REF1, l(3)j5D5, L Ascorbic Acid Peroxidase, Mass Spectrum Analysis, 24B, L-Ascorbic, Migrant Worker, Biologic Drugs, Man (Taxonomy), Analyses, Natural, developmental field, labeling., Nonmigrant, stru, ATAPX1, proteins, procedures, l(3)S053606, Biological Drugs, Transient, CG10293, Gaps, l(3)j5B5, Biological Medicine, LGP120, Methodological Studies, Squatters, Medicine, F24B9_2, Medicines, ascorbate peroxidase 1, Nerve Cell, Biologic Drug, 0904/17, Biologic Products, Ascorbate, Arts, Modern, 6-n-propylchromone-2-carboxylic acid, future organ, Procedure, nanometre, Spectrum Analysis, Spectroscopy, P2B, Lamp-1, Prgs, SZ1, Biopharmaceuticals, Migrant, Biologic Product, Genetic Materials, Workers, HAP1, Migrants and Transients, Genetic Material, Industrial, Nerve Cells, Industrial Arts, 120 kDa lysosomal membrane glycoprotein, Biological Medicines, Biological Drug, ASCORBATE PEROXIDASE, Spectrometry, L-Ascorbic Acid, Biologics, INSDC_feature:gene, Methodological, Methodological Study, human, APE1, Material, Cells, ATAPX01, Migrants, anon-EST:Liang-2.39, Cistron, APEX1, LGP-A, humans, APEN, lysosome membrane, human being, Procedures, Biologic Pharmaceuticals, Peptidomics, P62, apex, Nerve, Gene, Spectrum Analyses, protein-containing complex, Migrant Workers, Human, method, LAMPA, organ field, polypeptide chain, Homo sapiens, method used in an experiment, Studies, Gene Products, Mass, field, Technique, Man, APEX, Mass Spectroscopy, Biologicals, Drugs, study, l(3)s2612, Genetic, Acid Peroxidase, Transients, Natural Product, Nonmigrants, labeling, Worker, Peroxidase, Apex, Study, Squatter, CD107a, LGP-120, DmelCG10293, Neuron, Peroxidases, Biologic, Pharmaceuticals, Products, protein complex, clone 2.39, Biologic Medicines, Proteins, qkr, l(3)S090417, maternal effect embryo arrest 6, Cistrons, Cell, AI196048, MS, iPSC, native protein, natural protein, KH93F, Protein, Biopharmaceutical, Nomad, PCCA, techniques, Lysosomal membrane glycoprotein A, Mass Spectrum Analyses, L-Ascorbic Acid Peroxidase, nm, who, CS1, Mass Spectrum, Ascorbate Peroxidase, CD107 antigen-like family member A, Who/How, Lysosome-associated membrane protein 1, Protein Gene Products, Drug, plan specification, Gene Proteins, Ref-1, Nomads, Modern Man, qkr[93F], Natural Products, REF-1, APE, C79630, methodology0falseDevelopment and Comparative Evaluation of Endolysosomal Proximity Labeling-based Proteomic Methods in Human iPSC-derived NeuronsProximity-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. Sat Oct 10 07:08:00 BST 2020MSV00008626033201688