This study is supported by the NIH grant (R01NS121608). A.M.F. acknowledges the ARCS-Metro Washington Chapter Scholarship and the Bourbon F. Scribner Endowment Fellowship. We thank the Michael Ward lab at the National Institute for Neurological Disorders and Stroke (NINDS) for molecular biology support and the i3Neuron technology development.

<ol><li>De Duve, C., Wattiaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functions+of+lysosomes%5BTitle%5D)+AND+%22Annual+Review+of+Physiology%22%5BJournal%5D)">Functions of lysosomes.</a> Annual Review of Physiology. 28 (1), 435-492 (1966).</li>
<li>Ballabio, A., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosomes+as+dynamic+regulators+of+cell+and+organismal+homeostasis%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Lysosomes as dynamic regulators of cell and organismal homeostasis.</a> Nature Reviews Molecular Cell Biology. 21 (2), 101-118 (2020).</li>
<li>Lawrence, R. E., Zoncu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+lysosome+as+a+cellular+centre+for+signalling%2C+metabolism+and+quality+control%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">The lysosome as a cellular centre for signalling, metabolism and quality control.</a> Nature Cell Biology. 21 (2), 133-142 (2019).</li>
<li>Schröder, B. A., Wrocklage, C., Hasilik, A., Saftig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+proteome+of+lysosomes%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">The proteome of lysosomes.</a> Proteomics. 10 (22), 4053-4076 (2010).</li>
<li>Lie, P. P. Y., Nixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosome+trafficking+and+signaling+in+health+and+neurodegenerative+diseases%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">Lysosome trafficking and signaling in health and neurodegenerative diseases.</a> Neurobiology of Disease. 122, 94-105 (2019).</li>
<li>Leeman, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosome+activation+clears+aggregates+and+enhances+quiescent+neural+stem+cell+activation+during+aging%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging.</a> Science. 359 (6381), 1277-1283 (2018).</li>
<li>Wallings, R. L., Humble, S. W., Ward, M. E., Wade-Martins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosomal+dysfunction+at+the+centre+of+Parkinson%27s+disease+and+frontotemporal+dementia%2Famyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Trends+in+Neurosciences%22%5BJournal%5D)">Lysosomal dysfunction at the centre of Parkinson's disease and frontotemporal dementia/amyotrophic lateral sclerosis.</a> Trends in Neurosciences. 42 (12), 899-912 (2019).</li>
<li>Ferguson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+lysosomes%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Neuronal lysosomes.</a> Neuroscience Letters. 697, 1-9 (2019).</li>
<li>Rost, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+acidification+of+synaptic+vesicles+and+lysosomes%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Optogenetic acidification of synaptic vesicles and lysosomes.</a> Nature Neuroscience. 18 (12), 1845-1852 (2015).</li>
<li>Liao, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+granules+hitchhike+on+lysosomes+for+long-distance+transport%2C+using+annexin+A11+as+a+molecular+tether%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">RNA granules hitchhike on lysosomes for long-distance transport, using annexin A11 as a molecular tether.</a> Cell. 179 (1), 147-164 (2019).</li>
<li>Kuijpers, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+autophagy+regulates+presynaptic+neurotransmission+by+controlling+the+axonal+endoplasmic+reticulum%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neuronal autophagy regulates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum.</a> Neuron. 109 (2), 299-313 (2021).</li>
<li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+serotonin+neurons+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Generation of serotonin neurons from human pluripotent stem cells.</a> Nature Biotechnology. 34 (1), 89-94 (2016).</li>
<li>Dolmetsch, R., Geschwind, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+brain+in+a+dish%3A+The+promise+of+iPSC-derived+neurons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The human brain in a dish: The promise of iPSC-derived neurons.</a> Cell. 145 (6), 831-834 (2011).</li>
<li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scalable+production+of+iPSC-derived+human+neurons+to+identify+tau-lowering+compounds+by+high-content+screening%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Scalable production of iPSC-derived human neurons to identify tau-lowering compounds by high-content screening.</a> Stem Cell Reports. 9 (4), 1221-1233 (2017).</li>
<li>Fernandopulle, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcription+factor-mediated+differentiation+of+human+iPSCs+into+neurons%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">Transcription factor-mediated differentiation of human iPSCs into neurons.</a> Current Protocols in Cell Biology. 79 (1), 1-48 (2018).</li>
<li>Sunbul, M., Andres, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSunbul%2C+M+%22Proximity+labeling%3A+Methods+and+protocols%22">Proximity labeling: Methods and protocols</a>. , Humana Press. (2019).</li>
<li>Lam, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+evolution+of+APEX2+for+electron+microscopy+and+proximity+labeling%5BTitle%5D)+AND+%22Nature+methods%22%5BJournal%5D)">Directed evolution of APEX2 for electron microscopy and proximity labeling.</a> Nature methods. 12 (1), 51-54 (2015).</li>
<li>Rhee, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+mapping+of+mitochondria+in+living+cells+via+spatially+restricted+enzymatic+tagging%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.</a> Science. 339 (6125), 1328-1331 (2013).</li>
<li>Lobingier, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+approach+to+spatiotemporally+resolve+protein+interaction+networks+in+living+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">An approach to spatiotemporally resolve protein interaction networks in living cells.</a> Cell. 169 (2), 350-360 (2017).</li>
<li>Paek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multidimensional+tracking+of+GPCR+signaling+via+peroxidase-catalyzed+proximity+labeling%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling.</a> Cell. 169 (2), 338-349 (2017).</li>
<li>Fazal, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atlas+of+subcellular+RNA+localization+revealed+by+APEX-Seq%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Atlas of subcellular RNA localization revealed by APEX-Seq.</a> Cell. 178 (2), 473-490 (2019).</li>
<li>Frankenfield, A. M., Fernandopulle, M. S., Hasan, S., Ward, M. E., Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+comparative+evaluation+of+endolysosomal+proximity+labeling-based+proteomic+methods+in+human+iPSC-derived+neurons%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Development and comparative evaluation of endolysosomal proximity labeling-based proteomic methods in human iPSC-derived neurons.</a> Analytical Chemistry. 92 (23), 15437-15444 (2020).</li>
<li>Li, J., Pfeffer, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosomal+membrane+glycoproteins+bind+cholesterol+and+contribute+to+lysosomal+cholesterol+export%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export.</a> eLife. 5, 21635(2016).</li>
<li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+versatile+polypharmacology+platform+promotes+cytoprotection+and+viability+of+human+pluripotent+and+differentiated+cells%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.</a> Nature Methods. 18 (5), 528-541 (2021).</li>
<li>Li, H., Frankenfield, A. M., Houston, R., Sekine, S., Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thiol-cleavable+biotin+for+chemical+and+enzymatic+biotinylation+and+its+application+to+mitochondrial+TurboID+proteomics%5BTitle%5D)+AND+%22Journal+of+the+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">Thiol-cleavable biotin for chemical and enzymatic biotinylation and its application to mitochondrial TurboID proteomics.</a> Journal of the American Society for Mass Spectrometry. 32 (9), 2358-2365 (2021).</li>
<li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+proteomic+and+metabolomic+analyses+of+the+mitochondrial+neurodegenerative+disease+MELAS%5BTitle%5D)+AND+%22Molecular+Omics%22%5BJournal%5D)">Integrated proteomic and metabolomic analyses of the mitochondrial neurodegenerative disease MELAS.</a> Molecular Omics. 18 (3), 196-205 (2022).</li>
<li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MaxQuant+enables+high+peptide+identification+rates%2C+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nature Biotechnology. 26 (12), 1367-1372 (2008).</li>
<li>Kong, A. T., Leprevost, F. V., Avtonomov, D. M., Mellacheruvu, D., Nesvizhskii, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MSFragger%3A+Ultrafast+and+comprehensive+peptide+identification+in+mass+spectrometry-based+proteomics%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">MSFragger: Ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics.</a> Nature Methods. 14 (5), 513-520 (2017).</li>
<li>Frankenfield, A. M., Ni, J., Ahmed, M., Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+do+protein+contaminants+influence+DDA+and+DIA+proteomics%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">How do protein contaminants influence DDA and DIA proteomics.</a> bioRxiv. , (2022).</li>
<li>Kuleshov, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enrichr%3A+A+comprehensive+gene+set+enrichment+analysis+web+server+2016+update%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Enrichr: A comprehensive gene set enrichment analysis web server 2016 update.</a> Nucleic Acids Research. 44, 90-97 (2016).</li>
<li>Szklarczyk, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STRING+v11%3A+Protein-protein+association+networks+with+increased+coverage%2C+supporting+functional+discovery+in+genome-wide+experimental+datasets%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets.</a> Nucleic Acids Research. 47, 607-613 (2019).</li>
<li>Kissing, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+the+vacuolar-type+H%2B-ATPase+complex+in+liver+causes+MTORC1-independent+accumulation+of+autophagic+vacuoles+and+lysosomes%5BTitle%5D)+AND+%22Autophagy%22%5BJournal%5D)">Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.</a> Autophagy. 13 (4), 670-685 (2017).</li>
<li>kleine Balderhaar, H. J., Ungermann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CORVET+and+HOPS+tethering+complexes+-+coordinators+of+endosome+and+lysosome+fusion%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion.</a> Journal of Cell Science. 126 (6), 1307-1316 (2013).</li>
<li>Zhang, M., Chen, L., Wang, S., Wang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rab7%3A+Roles+in+membrane+trafficking+and+disease%5BTitle%5D)+AND+%22Bioscience+Reports%22%5BJournal%5D)">Rab7: Roles in membrane trafficking and disease.</a> Bioscience Reports. 29 (3), 193-209 (2009).</li>
<li>Cheng, X. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+LAMP1-labeled+nondegradative+lysosomal+and+endocytic+compartments+in+neurons%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Characterization of LAMP1-labeled nondegradative lysosomal and endocytic compartments in neurons.</a> Journal of Cell Biology. 217 (9), 3127-3139 (2018).</li>
<li>Eskelinen, E. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+LAMP-1+and+LAMP-2+in+lysosome+biogenesis+and+autophagy%5BTitle%5D)+AND+%22Molecular+Aspects+of+Medicine%22%5BJournal%5D)">Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy.</a> Molecular Aspects of Medicine. 27 (5-6), 495-502 (2006).</li>
<li>Sancak, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ragulator-rag+complex+targets+mTORC1+to+the+lysosomal+surface+and+is+necessary+for+its+activation+by+amino+acids%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Ragulator-rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids.</a> Cell. 141 (2), 290-303 (2010).</li>
<li>Abu-Remaileh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosomal+metabolomics+reveals+V-ATPase-+and+mTOR-dependent+regulation+of+amino+acid+efflux+from+lysosomes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.</a> Science. 358 (6364), 807-813 (2017).</li>
<li>Ong, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+isotope+labeling+by+amino+acids+in+cell+culture%2C+SILAC%2C+as+a+simple+and+accurate+approach+to+expression+proteomics%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Proteomics%22%5BJournal%5D)">Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.</a> Molecular & Cellular Proteomics. 1 (5), 376-386 (2002).</li>
<li>Tao, W. A., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+quantitative+proteomics+via+stable+isotope+tagging+and+mass+spectrometry%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.</a> Current Opinion in Biotechnology. 14 (1), 110-118 (2003).</li>
<li>Hao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+proteomic+analysis+of+a+genetically+induced+prostate+inflammation+mouse+model+via+custom+4-plex+DiLeu+isobaric+labeling%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Renal+Physiology%22%5BJournal%5D)">Quantitative proteomic analysis of a genetically induced prostate inflammation mouse model via custom 4-plex DiLeu isobaric labeling.</a> American Journal of Physiology - Renal Physiology. 316 (6), 1236-1243 (2019).</li>
<li>Thompson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tandem+mass+tags%3A+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS%2FMS%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.</a> Analytical Chemistry. 75 (8), 1895-1904 (2003).</li>
<li>Qin, W., Cho, K. F., Cavanagh, P. E., Ting, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deciphering+molecular+interactions+by+proximity+labeling%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Deciphering molecular interactions by proximity labeling.</a> Nature Methods. 18, 133-143 (2021).</li>
<li>Go, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+proximity-dependent+biotinylation+map+of+a+human+cell%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A proximity-dependent biotinylation map of a human cell.</a> Nature. 595 (7865), 120-124 (2021).</li>
</ol>

The authors declare no competing financial interests.

Using this LAMP1-APEX probe, proteins on and near the lysosomal membrane are biotinylated and enriched. Given the typical lysosome diameter of 100-1,200 nm, this method provides excellent intracellular resolution with a 10-20 nm labeling radius. LAMP1 is an abundant lysosomal membrane protein and a classical marker for lysosomes, serving as an excellent bait protein for lysosomal APEX labeling at the endogenous expression level. However, limitations also exist when using LAMP1 to target lysosomes, as LAMP1 is also present in late endosomes and nondegradative lysosomes35. Most lysosomal markers are also expressed in late endosomes, which eventually mature into lysosomes. Alternative bait proteins to target lysosomes are LAMPTOR, LAMP2, and TMEM19235,36,37. It is important to note that reactive biotin radicals do not penetrate the membrane. Therefore, most lysosomal lumen-only proteins are not captured in this LAMP1-APEX proteomics method. Lysosomal lumen proteins can be obtained by lysosomal isolation via the traditional gradient centrifugation method or lysosomal immunopurification4,38. However, proteins on the lysosomal membrane may be disrupted during lysosomal isolation and lose the information for transient and dynamic lysosomal interactions. Therefore, lysosomal proximity labeling and lysosomal isolation can be combined to obtain a complete snapshot of lysosomal activities both outside and inside lysosomes.
To minimize variability from iPSC-neuron culture, the neuron plating density must be consistent across all biological replicates and comparison groups. The same levels of neuronal maturation and health are also critical for this reason. During APEX activation, biotin-phenol and H2O2 addition to the cells must be conducted by prior mixing with warm culture medium and then adding the mixture to the cells, followed by immediate, gentle shaking to ensure even distribution. The use of H2O2 also raises concerns about oxidative stress and perturbations in the dynamic microenvironment of the cell. Although no significant changes were found at the protein abundance level, more peptides were modified with methionine oxidation in the H2O2-treated versus control neurons22. Therefore, strict control of the H2O2 activation time (1 min) is critical to minimize oxidative stress and reduce the diffusion of biotin cloud to ensure a specific labeling radius surrounding the bait protein.
For nonpolarized cell lines such as HEK and U2OS, cells can be harvested by pelleting after proximity labeling to remove the supernatant containing free biotin. However, neurons must be harvested by directly adding cell lysis buffer to the plate and scraping into tubes to avoid neurite damage and sample loss during pelleting. The presence of free biotin can saturate the streptavidin beads. The complete removal of free biotin can be achieved by multiple washes and incubation with quench buffer in neurons and/or protein precipitation after cell lysis. As different bait proteins have different expression levels, a dot blot assay needs to be conducted for each new APEX probe. Once the optimal beads/protein ratio is determined, the amount of starting protein and beads volume should be consistent for all replicates for the same APEX probe. When comparing different probes, such as LAMP1-APEX versus cytosolic-APEX, it is recommended to use the same volume of beads but vary the amount of starting protein to reflect the optimal beads/protein ratios for different APEX probes. To further reduce experimental variations and increase throughput, stable isotope labeling by amino acids in cell culture (SILAC) can be conducted39. Multiplexed isobaric labeling can also be used to chemically label peptides after protein digestion via TMT/iTRAQ/DiLeu tags20,40,41,42.
Proximity labeling has been widely applied to capture the cellular and molecular microenvironment in various organisms43. However, proximity labeling still faces many technical challenges, such as contamination from streptavidin signals, the use of hydrogen peroxide for enzyme activation, and the presence of endogenously biotinylated mitochondrial carboxylases. Therefore, proximity labeling proteomic experiments require careful planning and quality control. To help researchers troubleshoot their proximity labeling experiment, we provide a brief guide for common problems and solutions in Table 2. Most recently, a cleavable proximity labeling method was developed using thiol-cleavable biotin25. Biotinylated proteins can, therefore, be cleaved off beads using a reducing reagent such as TCEP without the need for on-beads digestion. This cleavable biotin method can dramatically reduce interference signals from streptavidin, endogenously biotinylated carboxylases, and nonspecific binding. Ongoing efforts will apply this cleavable biotin method to LAMP1-APEX proteomics to improve labeling specificity and accuracy. Proximity labeling probes can also be designed to target other subcellular compartments44. The amount and type of proteins identified are dependent on the nature of the bait protein, its intracellular environment, and the expression level of the proximity labeling probe. This endogenous LAMP1-APEX proteomics method provides a valuable tool to study the dynamic lysosomal activity in human neurons. The detailed protocol and methodology optimization are also applicable to other proximity labeling probes and chemical biotinylation, serving as a useful resource for the proteomics community.

Lysosomes are catabolic organelles that degrade macromolecules via the lysosomal-autophagy pathway1. Besides degradation, lysosomes are involved in diverse cellular functions such as signaling transduction, nutrient sensing, and secretion2,3,4. Perturbations in lysosomal function have been implicated in lysosomal storage disorders, cancer, aging, and neurodegeneration3,5,6,7. For postmitotic and highly polarized neurons, lysosomes play critical roles in neuronal cellular homeostasis, neurotransmitter release, and long-distance transport along the axons8,9,10,11. However, investigating lysosomes in human neurons has been a challenging task. Recent advancements in induced pluripotent stem cell (iPSC)-derived neuron technologies have enabled the culture of live human neurons that were previously inaccessible, bridging the gap between animal models and human patients to study the human brain12,13. Particularly, the advanced i3Neuron technology stably integrates the neurogenin-2 transcription factor into the iPSC genome under a doxycycline-inducible promoter, driving iPSCs to differentiate into pure cortical neurons in 2 weeks14,15.
Due to the highly dynamic lysosomal activity, capturing lysosomal interactions with other cellular components is technically challenging, particularly in a high-throughput fashion. Proximity labeling technology is well-suited to studying these dynamic interactions because of its capability to capture both stable and transient/weak protein interactions with exceptional spatial specificity16,17. Engineered peroxidase or biotin ligase can be genetically fused to the bait protein. Upon activation, highly reactive biotin radicals are produced to covalently label neighboring proteins, which can then be enriched by streptavidin-coated beads for downstream bottom-up proteomics via liquid chromatography-mass spectrometry (LC-MS) platforms17,18,19,20,21.
An endogenous lysosomal proximity labeling proteomics method was recently developed to capture the dynamic lysosomal microenvironment in i3Neurons22. Engineered ascorbate peroxidase (APEX2) was knocked-in on the C-terminus of the lysosomal associated membrane protein 1 (LAMP1) in iPSCs, which can then be differentiated into cortical neurons. LAMP1 is an abundant lysosomal membrane protein and a classical lysosomal marker23. LAMP1 is also expressed in late endosomes, which mature into lysosomes; these late endosome-lysosomes and nondegradative lysosomes are all referred to as lysosomes in this protocol. This endogenous LAMP1-APEX probe, expressed at the physiological level, can reduce LAMP1 mislocalization and overexpression artifacts. Hundreds of lysosomal membrane proteins and lysosomal interactors can be identified and quantified with excellent spatial resolution in live human neurons.
Here, a detailed protocol for lysosome proximity labeling proteomics in human iPSC-derived neurons is described with further improvements from the recently published method22. The overall workflow is illustrated in Figure 1. The protocol includes hiPSC-derived neuron culture, proximity labeling activation in neurons, validation of APEX activity by fluorescence microscopy, determination of an optimal streptavidin beads-to-input protein ratio, enrichment of biotinylated proteins, on-beads protein digestion, peptide desalting and quantification, LC-MS analysis, and proteomics data analysis. Troubleshooting guidelines and experimental optimizations are also discussed to improve proximity labeling quality control and performance.

<table><tbody><tr><td>10% (w/v) Saponin solution</td><td>Acros Organics</td><td>419231000</td><td>Flourescent Microscopy</td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A1110501</td><td>cell detachment solution, Cell Culture</td></tr><tr><td>B27 Supplement</td><td>Fisher Scientific</td><td>17504044</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>BDNF</td><td>PeproTech</td><td>450-02</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B6768</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A8806</td><td>To make standard solutions to measure total protein concentrations</td></tr><tr><td>Brainphys neuronal medium</td><td>STEMCELL Technologies</td><td>5790</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>CD45R (B220) Antibody Alexa Fluor 561</td><td>Thermo Fisher Scientific</td><td>505-0452-82</td><td>Flourescent Microscopy</td></tr><tr><td>Chroman1 ROCK inhibitor</td><td>Tocris</td><td>716310</td><td>Cell Culture</td></tr><tr><td>cOmplete mini Protease Inhibitor</td><td>Roche</td><td>4693123001</td><td>cocktail inhibitor in Lysis Buffer</td></tr><tr><td>DC Protein Assay Kit II</td><td>Bio-Rad</td><td>5000112</td><td>To determine total protein concentrations of cell lysate</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Proximity-labeling Reaction</td></tr><tr><td>DMEM/F12 medium</td><td>Thermo Fisher Scientific</td><td>11320082</td><td>Cell Culture, Dish Coating</td></tr><tr><td>DMEM/F12 medium with HEPES</td><td>Thermo Fisher Scientific</td><td>11330057</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td>Flourescent Microscopy</td></tr><tr><td>Doxycycline hyclate, &amp;ge;98% (HPLC)</td><td>Sigma-Aldrich</td><td>D9891-1G</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Essential 8 Medium</td><td>Thermo Fisher Scientific</td><td>A1517001</td><td>Cell Culture</td></tr><tr><td>Essential 8 Supplement (50x)</td><td>Thermo Fisher Scientific</td><td>A1517101</td><td>Cell Culture</td></tr><tr><td>Extraction plate vacuum manifold kit</td><td>Waters</td><td>WAT097944</td><td>For Peptide desalting</td></tr><tr><td>Formic Acid (FA)</td><td>Fisher Scientific</td><td>A11750</td><td>For LC-MS analysis</td></tr><tr><td>GDNF</td><td>PeproTech</td><td>450-10</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>Hoechst dye</td><td>Thermo Fisher Scientific</td><td>62239</td><td>Flourescent Microscopy</td></tr><tr><td>HPLC grade methanol</td><td>Fisher Scientific</td><td>A452</td><td>For Peptide desalting</td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W5</td><td>For Peptide desalting</td></tr><tr><td>Human induced pluripotent stem cells</td><td>Corriell Institute</td><td>GM25256</td><td>Cell Culture</td></tr><tr><td>Hydrogen peroxide, ACS, 29-32% w/w aq. soln., stab.</td><td>Thermo Fisher Scientific</td><td>AA33323AD</td><td>Proximity-labeling Reaction</td></tr><tr><td>Iodoacetamide (IAA)</td><td>Millipore Sigma</td><td>I6125</td><td>For Protein Digestion</td></tr><tr><td>Laminin</td><td>Fisher Scientific</td><td>23017015</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>LC-MS grade Acetonitrile</td><td>Fisher Scientific</td><td>A955</td><td>For LC-MS analysis</td></tr><tr><td>LC-MS grade water</td><td>Fisher Scientific</td><td>W64</td><td>For LC-MS analysis</td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>25-030-081</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Matrigel</td><td>Thermo Fisher Scientific</td><td>08-774-552</td><td>basement membrane matrix, Cell Culture, Dish Coating</td></tr><tr><td>Mouse anti-human LAMP1 monoclonal antibody</td><td>Developmental Studies Hybridoma Bank</td><td>h4a3</td><td>Flourescent Microscopy</td></tr><tr><td>N-2 Supplement (100x)</td><td>Fisher Scientific</td><td>17-502-048</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Nitrocellulose Membrane, Precut, 0.45 &amp;micro;m, 7 x 8.5 cm</td><td>Bio-Rad</td><td>1620145</td><td>To conduct dot blot assay for bead titration</td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Fisher Scientific</td><td>11-140-050</td><td>Cell Culture, Induction Medium</td></tr><tr><td>NT-3</td><td>PeproTech</td><td>450-03</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>Oasis HLB 96-well solid phase extraction plate</td><td>Waters</td><td>186000309</td><td>For Peptide desalting</td></tr><tr><td>Odyssey Blocking Buffer (TBS)</td><td>LI-COR Biosciences</td><td>927-50000</td><td>To conduct dot blot assay for bead titration</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Flourescent Microscopy</td></tr><tr><td>Phenol Biotin (1,000x stock)</td><td>Adipogen</td><td>41994-02-9</td><td>Proximity-labeling Reaction</td></tr><tr><td>Phosphate-buffered saline (PBS) without calcium or magnesium</td><td>Gibco</td><td>10010049</td><td>Cell Culture, Proximity-labeling Reaction, Flourescent Microscopy</td></tr><tr><td>Pierce Quantitative Colorimetric Peptide Assay</td><td>Thermo Fisher</td><td>23275</td><td>Peptide Concentration Assay</td></tr><tr><td>Poly-L-Ornithine (PLO)</td><td>Millipore Sigma</td><td>P3655</td><td>Cell Culture, Dish Coating</td></tr><tr><td>Sodium Ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td>Proximity-Labeling Quench Buffer, Lysis Buffer</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td>Proximity-Labeling Quench Buffer, Lysis Buffer, Flourescent Microscopy</td></tr><tr><td>Sodium chloride</td><td>Thermo Fisher Scientific</td><td>S271500</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Thermo Fisher Scientific</td><td>BP1311220</td><td>Lysis Buffer, Dot blot assay buffer, Beads wash buffer</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>415413</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>Sodium tetraborate</td><td>Sigma-Aldrich</td><td>221732</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>SpeedVac concentrator</td><td></td><td></td><td>vacuum concentrator</td></tr><tr><td>Streptavidin Magnetic Sepharose Beads</td><td>Cytiva (formal GE)</td><td>28-9857-99</td><td>Enrich biotinylated proteins</td></tr><tr><td>Streptavidin, Alexa Fluor 680 Conjugate</td><td>Thermo Fisher Scientific</td><td>S32358</td><td>To conduct dot blot assay for bead titration</td></tr><tr><td>Thermomixer</td><td></td><td></td><td>temperature-controlled mixer</td></tr><tr><td>Trifluoacetic acid (TFA)</td><td>Millipore Sigma</td><td>302031</td><td>For Peptide desalting</td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Millipore Sigma</td><td>C4706</td><td>For Protein Digestion</td></tr><tr><td>Tris-HCl</td><td>Thermo Fisher Scientific</td><td>BP152500</td><td>Lysis Buffer, Dot blot assay buffer, Beads wash buffer</td></tr><tr><td>Triton-X</td><td>Thermo Fisher Scientific</td><td>BP151500</td><td>Beads wash buffer</td></tr><tr><td>TROLOX</td><td>Sigma-Aldrich</td><td>648471</td><td>Proximity-Labeling Quench Buffer, Lysis Buffer</td></tr><tr><td>Trypsin/Lys-C Mix, Mass Spec Grade</td><td>Promega</td><td>V5073</td><td>For Protein Digestion</td></tr><tr><td>TWEEN&amp;nbsp;20</td><td>Millipore Sigma</td><td>P1379</td><td>Dot blot assay buffer</td></tr><tr><td>Urea</td><td>Thermo Fisher Scientific</td><td>BP169500</td><td>Beads wash and On-Beads Digestion Buffer</td></tr><tr><td>Vitronectin</td><td>STEMCELL Technologies</td><td>7180</td><td>Cell Culture, Dish Coating</td></tr><tr><td>Y-27632 ROCK inhibitor</td><td>Selleck</td><td>S1049</td><td>Cell Culture</td></tr></tbody></table>

characterization of neuronal lysosome interactome with proximity labeling proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

This lysosome proximity labeling proteomics study was conducted in human iPSC-derived neurons to capture the dynamic lysosomal microenvironment in situ in live neurons. Cell morphologies of hiPSCs and hiPSC-derived neurons at different time points are illustrated in Figure 2A. Human iPSCs grow in colonies in E8 medium. Differentiation is initiated by plating iPSCs into doxycycline-containing neuron induction medium. Neurite extensions become more visible each day during the 3 day differentiation. After switching to PLO-coated plates in neuron medium, the neurites form a network between neurons, and axonal extensions become more visible as the neurons reach maturation in 2 weeks. In i3Neurons, the localization of the APEX probe is validated by fluorescence microscopy following rapid APEX activation. Biotinylated proteins are stained using streptavidin (SA) antibody, and lysosomes are stained using anti-LAMP1 antibody. The merged image validates the correct localization of LAMP1-APEX to the bait protein (Figure 2B).
The bead-titration assay is crucial to determine the optimal beads-to-protein ratio so that the amount of streptavidin beads is enough to enrich all the biotinylated proteins but not so excessive to cause serious streptavidin contamination in LC-MS. The optimal volume of beads needed for 50 &#181;g of input protein sample is selected on the basis of where the exponential decay of the curve ends (Figure 3A) As shown in Figure 3A, dot-blot signals from the beads-protein incubation supernatant decreased as more biotinylated proteins were captured with increased amounts of the streptavidin beads. For endogenous LAMP1-APEX samples, 5 &#181;L of streptavidin beads were optimal for 50 &#181;g of input protein (highlighted in Figure 3A). Following enrichment, the amount of protein captured by the streptavidin beads is unknown. Excess proteolytic enzyme (trypsin) can increase enzyme autodigestion, with abundant trypsin peptide peaks in LC-MS. Excessive trypsin can also digest more streptavidin peptides in the samples. Therefore, the amount of protease needed for on-beads digestion should be optimized. Compared to trypsin alone, on-beads digestion with Trypsin/Lys-C mix resulted in the identification of more proteins and peptides and fewer missed cleavages (Figure 3B). Additionally, 1-1.5 &#181;g of protease per 250 &#181;L of streptavidin magnetic beads was optimal to obtain the highest number of identified proteins and the lowest percentage of missed cleavages (Figure 3C). With an optimal beads-to-protein ratio, the same amount of beads should capture the same amount of biotinylated proteins. Therefore, this optimized protease amount can be used for all experiments that enrich biotinylated proteins using the same streptavidin magnetic beads.
Peroxidase-based proximity labeling enzymes are activated by biotin-phenol incubation and brief H2O2 treatment (1 min). This step is a major source of variation in proximity-labeling proteomics. We previously found that normalization to the most abundant, endogenously biotinylated carboxylase, PCCA, can significantly reduce experimental variations, allowing the comparison of proximity labeling proteomics data across different experimental batches (Figure 4)22. For endogenous LAMP1-APEX neurons, the parental line without LAMP1-APEX probe expression was used as a control group. Control neurons were also treated with biotin-phenol and H2O2. The distribution of protein ratios of LAMP1-APEX versus the control group is illustrated in Figure 5A. All the endogenously biotinylated carboxylases were enriched by streptavidin-coated beads but remained unchanged. As shown in the GO-term analysis and protein network analysis (Figure 5B,C), both stable lysosomal membrane proteins and transient lysosomal interactors related to endolysosomal trafficking and transport were enriched in LAMP1-APEX proteomics32,33,34.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64132/64132fig01.jpg" /> 
Figure 1: Overall workflow for lysosome proximity labeling proteomics in hiPSC-derived neurons. Abbreviations: hiPSC = human induced pluripotent stem cell; LAMP1 = lysosomal associated membrane protein 1; APEX = ascorbate peroxidase; dox = doxycycline; BP = biotin-phenol; DCA = detergent-compatible protein assay; SA = streptavidin; LC-MS/MS = liquid chromatography-tandem mass spectrometry; PCCA = propionyl-CoA carboxylase, an endogenously biotinylated protein. <a href="https://www.jove.com/files/ftp_upload/64132/64132fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64132/64132fig02.jpg" /> 
Figure 2: Microscopic imaging of hiPSC-derived neurons and LAMP1-APEX activity. (A) Brightfield microscopy images of different stages of hiPSCs and hiPSC-derived neurons. (B) Fluorescence imaging of LAMP1-APEX activity in the neuron. Biotinylated signals stained against streptavidin colocalize with LAMP1 staining outside the nucleus (HOECHST). Scale bars = (A) 50 &#181;m, (B) 1 &#181;m. Abbreviations: hiPSC = human induced pluripotent stem cell; LAMP1 = lysosomal associated membrane protein 1; APEX = ascorbate peroxidase; SA = streptavidin. <a href="https://www.jove.com/files/ftp_upload/64132/64132fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64132/64132fig03.jpg" /> 
Figure 3: Optimization of beads-to-input protein ratio and enzymatic protein digestion can improve protein identifications and reduce interference. (A) Example of beads-titration assay results from dot-blot assay using 50 &#181;g of input protein and different amounts of streptavidin beads. (B) Trypsin/Lys-C mix resulted in better protein/peptide identification and fewer missed cleavages than trypsin alone. (C) Optimization of the amount of Trypsin/Lys-C for on-beads digestion. This figure has been modified from Frankenfield et al.22. <a href="https://www.jove.com/files/ftp_upload/64132/64132fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64132/64132fig04.jpg" /> 
Figure 4: Normalization of proximity labeling proteomics data to an endogenously biotinylated carboxylase, PCCA, can reduce quantification variations among biological replicates. This figure has been modified from Frankenfield et al.22. Abbreviation: PCCA = propionyl-CoA carboxylase. <a href="https://www.jove.com/files/ftp_upload/64132/64132fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64132/64132fig05.jpg" /> 
Figure 5: Lysosome proximity labeling proteomics enriched lysosomal membrane proteins and lysosomal interacting proteins in neurons. (A) Scatter plot of protein abundance ratios of LAMP1-APEX versus no APEX control showing enriched lysosomal membrane proteins and unchanged endogenously biotinylated proteins. (B) GO-term analysis of proteomics results proving enriched cellular component at the lysosome. (C) STRING protein network analysis showing that proteins directly interact with the bait protein (LAMP1), lysosomal membrane proteins, and lysosomal interactors such as membrane trafficking proteins. This figure has been modified from Frankenfield et al.22. Abbreviations: LAMP1 = lysosomal associated membrane protein 1; APEX = ascorbate peroxidase; GO = gene ontology. <a href="https://www.jove.com/files/ftp_upload/64132/64132fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium/Buffer</td>
			<td colspan="3">Component</td>
			<td>Protocol</td>
		</tr>
		<tr>
			<td>Basement membrane matrix (Matrigel) coating solution</td>
			<td colspan="3">1% basement membrane matrix stock, 99% DMEM/F12 medium</td>
			<td>1.1, 1.2</td>
		</tr>
		<tr>
			<td>Vitronectin coating solution</td>
			<td colspan="3">5 &#956;g/mL final concentration in PBS</td>
			<td>1.1</td>
		</tr>
		<tr>
			<td>E8 complete medium with ROCK inhibitor</td>
			<td colspan="3">98% E8 medium, 2% E8 supplement, 10 &#181;M Y-27632 or 50 nM Chroman1</td>
			<td>1.1</td>
		</tr>
		<tr>
			<td>Neuron Induction medium</td>
			<td colspan="3">97% DMEM/F12 with HEPES, 1% N2 supplement, 1% non-essential amino acids (NEAA), 1% L-glutamine, 2 &#956;g/mL doxycycline and ROCK inhibitor (10 &#956;M Y-27632 or 5 nM Chroman 1)</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>Neuron PLO coating solution</td>
			<td colspan="3">0.1 mg/mL Poly-L-Ornithine (PLO), 100 mM boric acid, 25 mM sodium tetraborate, 75 mM sodium chloride, 1 M sodium hydroxide</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>Neuron medium</td>
			<td colspan="3">98% cortical neuron medium, 2% B27 supplement, 10 ng/mL brain-derived neurotrophic factor (BDNF), 10 ng/mL glial-derived neurotrophic factor (GDNF), 10 ng/mL NT-3, 0.2 &#181;g/mL Laminin, 2 &#956;g/mL doxycycline</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>Quench buffer</td>
			<td colspan="3">10 mM sodium azide, 10 mM sodium ascorbate, 5 mM TROLOX in PBS</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>Cell lysis buffer</td>
			<td colspan="3">50 mM Tris-HCl, 500 mM NaCl, 0.2% SDS, 1% Triton, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 10 mM sodium azide, 10 mM sodium ascorbate, 5 mM TROLOX, protease inhibitor cocktail</td>
			<td>2.3</td>
		</tr>
		<tr>
			<td>TBS-T</td>
			<td colspan="3">0.05% Tween20, 20 mM Tris, 150 mM NaCl (pH 7.5)</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td colspan="3">2% SDS buffer</td>
			<td>5.2</td>
		</tr>
		<tr>
			<td>Buffer B</td>
			<td colspan="3">50 mM Tris-HCl, 500 mM NaCl, 2% Triton-X</td>
			<td>5.6</td>
		</tr>
		<tr>
			<td>Buffer C</td>
			<td colspan="3">50 mM Tris-HCl, 250 mM NaCl, 0.5% SDS, 0.5% Triton-X</td>
			<td>5.6</td>
		</tr>
		<tr>
			<td>Buffer D</td>
			<td colspan="3">2 M Urea, 50 mM Tris-HCl</td>
			<td>5.6</td>
		</tr>
	</tbody>
</table>
Table 1: Compositions of media and buffers used in this protocol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Problem</td>
			<td>Protocol</td>
			<td colspan="4">Solution/Suggestion</td>
		</tr>
		<tr>
			<td colspan="2">iPSC culture peeling off the plate</td>
			<td>1.1</td>
			<td colspan="4">Increase vitronectin coating concentration or time.</td>
		</tr>
		<tr>
			<td colspan="2">Some iPSCs did not differentiate into neurons</td>
			<td>1.2</td>
			<td colspan="4">Increase cell detachment solution treatment time to completely dissociate iPSCs during d0 differentitation.</td>
		</tr>
		<tr>
			<td colspan="2">Neuron culture peeling off the plate</td>
			<td>1.3</td>
			<td colspan="4">Washing and changing the medium must be gentle and from the side wall of the plate.</td>
		</tr>
		<tr>
			<td colspan="2">Proteins do not dissolve completely after acetone precipitation</td>
			<td>2.7</td>
			<td colspan="4">Reduce drying time of the protein pellet. Increase the volume of lysis buffer and sonicate briefly to help dissolution.</td>
		</tr>
		<tr>
			<td colspan="2">Weak streptavidin staining signals</td>
			<td>3</td>
			<td colspan="4">Increase H2O2 treatment time to be 2-3 s and swirl the plate for even distribution.</td>
		</tr>
		<tr>
			<td colspan="2">Low signal of beads titration assay</td>
			<td>4.2</td>
			<td colspan="4">Wait until membrane is completely dried and add more supernatant to the same spot (can repeat up to 3x) to enhance the signal intensity.</td>
		</tr>
		<tr>
			<td colspan="2">Magnetic beads not pelleting towards magnetic rack</td>
			<td>5</td>
			<td colspan="4">Magnetic bead mobility decreases in non-detergent containing buffer. Higher urea concentration up to 4 M or LC-MS-compatible detergent can be used in wash buffer D.</td>
		</tr>
		<tr>
			<td colspan="2">Magnetic beads loss during beads wash</td>
			<td>5</td>
			<td colspan="4">Increase the waiting time when sample tubes are placed on the magnetic beads (1 min or longer) before taking supernatant from the tubes.</td>
		</tr>
		<tr>
			<td colspan="2">Singly charged contamination peaks in LC-MS</td>
			<td>6</td>
			<td colspan="4">Peptide cleanup not sufficient. Increase washing volumes and times during peptide desalting.</td>
		</tr>
		<tr>
			<td colspan="2">Peptide assay low signal</td>
			<td>7</td>
			<td colspan="4">Resuspend peptide samples in lower volume to increase peptide concentration.</td>
		</tr>
		<tr>
			<td colspan="2">Overwhelming streptavidin signals in LC-MS</td>
			<td>8</td>
			<td colspan="4">Reduce the amount of streptavidin beads. If trypsin peak is also abundant, reduce trypsin amount.</td>
		</tr>
		<tr>
			<td colspan="2">Too many nonspecific labeling background</td>
			<td>9</td>
			<td colspan="4">Streptavidin beads wash was not sufficient. Remove all residual liquid during each washing step. Increase the time and volume for beads wash.</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting problems and solutions.

Watch this Scientific Journal Video about Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics at JoVE.com

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

characterization-neuronal-lysosome-interactome-with-proximity

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.4K Views.  The George Washington University. A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Video: Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biochemistry

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Biology

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Summary

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Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays