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Method Article

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

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DOI:

10.3791/64132

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June 23rd, 2022

 ,  , 
1Department of Chemistry, The George Washington University

* These authors contributed equally

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

All procedures were approved by the George Washington University biosafety and ethics committee. The compositions of media and buffers used in this protocol are provided in Table 1. The commercial product information used here is provided in the Table of Materials.

1. Human iPSC-derived neuron culture

  1. Human iPSC culture and LAMP1-APEX probe integration (7 days)
    1. Thaw Matrigel stock solution in an ice bucket at 4 °C overnight, aliquot 500 µL of the solution into cold sterile tubes, and store the aliquots at −80°C. Prepare 50 mL of coating solution by adding 500 µL of the Matrigel stock into 49.5 mL of cold DMEM/F12 medium.
      NOTE: Keep the Matrigel cold and use prechilled conical tubes and pipette tips to prevent polymerization.Coating solution can be stored at 4 °C for 2 weeks.
    2. Coat a 10 cm cell culture dish with 4 mL of coating solution for 1 h in a 37 °C incubator.
      NOTE: Vitronectin can be an alternative coating solution at 5 µg/mL concentration and 2 h coating for iPSC culture. Vitronectin (single protein component) is more expensive than Matrigel but has less batch variations and cleaner background signals than Matrigel, which is extracted from mouse tumor with numerous extracellular matrix proteins. Non-treated tissue culture plate is needed for vitronectin coating. Matrigel coating works for both treated and non-treated tissue culture plates.
    3. Thaw and plate 1-3 million hiPSCs on each coated 10 cm dish preloaded with 8 mL of Essential E8 complete medium supplemented with ROCK inhibitor (final concentration 10 µM Y-27632 or 50 nM Chroman1).
      NOTE: Adding ROCK inhibitor (Y-27632 or Chroman1) to the stem cell culture medium minimizes dissociation-induced apoptosis after splitting and cryogenic preservation. Chroman1 has recently been shown to be more potent than Y-27632 at inhibiting both ROCK1 and ROCK224.
    4. Change to 10 mL of E8 complete medium without ROCK inhibitor after the iPSCs form colonies, typically after 1-2 days. Maintain the iPSC culture in E8 complete medium and replace the supernatant with fresh medium every other day.
    5. Integrate the proximity labeling enzyme, engineered ascorbate peroxidase (APEX2), into the C-terminus of endogenous LAMP1 gene by CRISPR genome engineering. For detailed steps to generate an engineered stable cell line, refer to the previously published method (Frankenfield et al).22.
  2. Human iPSC-neuron differentiation (3 days)
    1. Coat a 10 cm cell culture dish overnight with 4 mL of Matrigel coating solution in a 37 °C incubator before differentiation.
    2. Maintain hiPSCs until ~70% confluence. Gently wash the hiPSCs with phosphate-buffered saline (PBS) 2x to remove dead cells. Aspirate the PBS and add3 mL of Accutase to the 10 cm dish of cells. Shake the plate for even distribution and incubate for 8 min in a 37 °C incubator.
    3. Add 2 mL of PBS to wash and lift the cells from the plate and collect all cell solution in a 15 mL conical tube. Pellet the cells by centrifugation at 300 × g for 5 min. Plate 2-4 × 106 hiPSCs onto a Matrigel-coated plate preloaded with 8 mL of warm neuron induction medium (Table 1). Evenly distribute the cells and place in a 37 °C incubator.
      NOTE: Neuron differentiation is driven by a doxycycline-inducible transcription factor, neurogenin-2 (NGN2), which was overexpressed in this stable hiPSC cell line15.
    4. Wash hiPSCs gently with PBS on day 1 of differentiation to remove dead cell debris. Replace with 10 mL of warm induction medium without ROCK inhibitor.
    5. Change with warm induction medium without ROCK inhibitor every day until day 3 of differentiation.
      NOTE: Neurons are ready to be replated into neuronal medium.
  3. Plating neurons and maintaining neuron culture (10 days)
    1. Prepare 0.1 mg/mL Poly-L-Ornithine(PLO) coating solution in Borate buffer (Table 1).
    2. Coat a cell culture dish with the PLO coating solution in a 37 °C incubator for at least 1 h or overnight prior to plating day 3 neurons. Wash the dish with 4 mL of sterile water three times and let it air dry completely inside the biosafety cabinet.
    3. Dissociate day 3 neurons from each 10 cm dish with 3 mL of Accutase and plate 8-10 million cells onto each 10 cm PLO-coated dish preloaded with warm cortical neuron medium (Table 1).
    4. Perform a half-medium change every 2-3 days with warm neuron medium until the i3Neurons reach maturation in 2 weeks after differentiation.

2. In situ proximity labeling and neuron lysis (2 h)

  1. Prepare 500 mM biotin-phenol (BP) stock solution in dimethyl sulfoxide (DMSO) and store at −20 °C. On the day of proximity labeling, dilute the BP stock solution with warm neuron medium and treat the neurons at a final concentration of 500 µM for 30 min in a 37 °C incubator.
    NOTE: Doxycycline is added in the neuron medium, which will activate APEX enzyme expression. Doxcycline needs to be added at least 24 h before the proximity labeling experiment.
  2. Initiate the labeling reaction by adding H2O2 at a final concentration of 1 mM into the neuron culture and incubate for exactly 1 min. Immediately aspirate the medium and rinse 3 times with quench buffer (Table 1).
    NOTE: H2O2 solution should be made fresh before the labeling reaction. The H2O2 activation needs to be exactly 1 min to reduce experimental variation and minimize oxidative stress caused by prolonged H2O2 treatment.
  3. Tilt the plate to aspirate all the residual buffer. Add ice-cold cell lysis buffer (Table 1) directly onto the neuron plate. Use 100 µL of cell lysis buffer per 1 million neurons. Swirl the plate for sufficient cell lysis and place on ice.
  4. Scrape the cell lysates into cold 1.5 mL tubes. Sonicate in an ice-cold water bath using a bath sonicator (>100 W) for 15 min with alternating 40 s on, 20 s off cycles.
    NOTE: Sufficiently lysed protein solution should be clear without pellets or excessive bubbles. Sufficient sonication of cell lysate is crucial to shear nucleic acids and reduce the stickiness of the cell lysate to reduce nonspecific binding in downstream procedures. A probe sonicator can also be used to provide stronger and faster cell lysis, but washing the probe between samples is required to minimize cross-contamination and sample loss.
  5. Add cold acetone (−20 °C) to the sample at a 4-fold volume of lysis buffer. Vortex briefly and incubate at −20 °C for 3 h.
  6. Centrifuge at 16,500 × g for 10 min at 2 °C. Remove the supernatant without disturbing the protein pellet. Wash the pellet with 1 mL of −20 °C acetone 2x and remove the supernatant after centrifugation.
  7. Dry the protein pellet with a vacuum concentrator for 1 min. Add cell lysis buffer to completely dissolve the pellet. Sonicate briefly if needed. Store the samples at −80 °C.
    ​NOTE: Acetone precipitation can remove residue biotin-phenol in the sample, which can interfere with downstream enrichment of biotinylated proteins.

3. Fluorescence microscopy to validate APEX localization and activity (1.5 days)

  1. Incubate the i3Neurons expressing endogenous LAMP1-APEX with 500 µM BP for 30 min at 37 °C. Activate the labeling with 1 mM H2O2 treatment for just 1 s to limit the diffusion of biotin cloud.
  2. Fix the cells immediately with 4% paraformaldehyde in the quench buffer and gently wash 3x with PBS.
    NOTE: For a 10 cm dish, 4-6 mL is needed for a sufficient wash.
  3. Block and permeabilize the cells using 3% donkey serum and 0.1% saponin in PBS for 30 min at room temperature (RT).
  4. Incubate the cells with primary anti-LAMP1 antibody (mouse monoclonal H3A4, 1:1,000) overnight at 4 °C.
  5. Wash the neurons 3 x gently with PBS. Incubate the neurons with anti-mouse AF561 secondary antibody (1:1,000) and Streptavidin-680 (1:1,000) for 1 h at RT.
  6. Wash 2x with PBS and incubate with Hoechst or 4',4-diamidino-2-phenylindole (DAPI) nuclear marker for 10 min at RT. Rinse the cells 2x with PBS.
  7. Visualize the fixed neurons under a fluorescence microscope. Observe biotinylated proteins stained with streptavidin and colocalized with LAMP1 staining outside the nucleus to validate the correct location of APEX activity.

4. Determining the streptavidin beads-to-input protein ratio (1.5 days)

  1. Perform detergent-compatible protein assay (DCA) to determine total protein concentrations in cell lysates
    1. Dissolve bovine serum albumin (BSA) protein standard in the cell lysis buffer at a concentration of 4 mg/mL. Prepare a series of BSA protein standard solutions (0.2-2 mg/mL, 5 dilutions) using the cell lysis buffer.
    2. Transfer 5 µL from each protein sample, BSA protein standard solution, and blank cell lysis buffer in triplicate into each well in a 96-well plate.
    3. Add 2 µL of reagent S per 1 mL of reagent A to get reagent A'. Add 25 µL of reagent A' to each well. Add 200 µL of reagent B to each well. Mix and pop bubbles if present.
    4. Incubate that 96-well plate at RT for 15 min, read the absorbance at 750 nm in a microplate reader, and quantify the concentrations of protein samples based on the BSA standard curve.
  2. Beads titration dot blot assay (1.5 days)
    1. Transfer a series of streptavidin magnetic beads slurry (20 µL, 15 µL, 12 µL, 10 µL, 8 µL, 4 µL, 1 µL, 0 µL) to PCR strip tubes. Put the PCR strip tubes on a magnetic rack, wash the beads 3x with 100 µL of 2% SDS buffer, and remove the wash buffer completely while on the magnetic rack.
    2. Add 50 µg of protein sample to each tube. Add more lysis buffer to a total volume of 80 µL. Close each tube tightly and rotate at 4 °C overnight.
    3. Centrifuge the PCR strip tubes briefly on a benchtop microcentrifuge. Place the PCR strip tubes on a magnetic rack for 1 min. Spot 2 µL of supernatant from each tube onto a dry nitrocellulose membrane and allow the membrane to dry completely.
      NOTE: If the signal intensity is low, more than 2 µL can be spotted onto the membrane. The volume can be increased by spotting several times on the same spot after the membrane is air-dried between each time. A Bio-Dot Apparatus can also be used.
    4. Incubate the membrane in blocking buffer (TBS) for 1 h. Incubate the membrane in Streptavidin Alexa Fluor 680 conjugate (1:1,000 in blocking buffer) for 1 h. Then, wash the membrane with TBS-T (Table 1) 5x.
      NOTE: An alternative to the dot blot assay is western blotting using a streptavidin antibody.
    5. Measure the fluorescent signal of each dot on the membrane under 680 nm wavelength, and generate a scatter plot with fluorescent signal over the volume of beads. Select the optimal volume of beads needed for 50 µg of input protein sample based on where the exponential decay of the curve ends .
      ​NOTE: Fluorescence intensity represents the abundance of biotinylated proteins in the supernatant, which should decrease with increasing amounts of beads.

5. Enriching biotinylated proteins and on-beads digestion (3 days)

  1. Transfer protein lysate samples from −80 °C and sonicate the samples in 1.5 mL tubes for 30 s in a bath sonicator to quickly thaw the solutions. Vortex and place the sample tubes on ice.
  2. Transfer 250 µL of streptavidin (SA) magnetic beads slurry to each 1.5 mL tube. Put the tubes on a magnetic rack, wash the beads 3x with 1 mL of Wash Buffer A (2% SDS), and remove the residual buffer.
  3. Based on the results of the dot blot assay and DC protein assay, calculate the amount of protein sample (µg) needed for 250 µL of the streptavidin magnetic beads slurry.
    NOTE: In this endogenously expressed LAMP1-APEX probe, 5 µL of beads are needed for 50 µg of input protein. Therefore, 2.5 mg of total protein is added to 250 µL of beads. Less than 250 µL of SA beads may be used for limited input sample material.
  4. Add cell lysis buffer to the tube containing the magnetic bead-lysate mixture to a total volume of 1 mL and rotate at 4 °C overnight.
    NOTE: Samples from different groups or replicates should be normalized to the same protein concentration, volume, and amount of beads to reduce experimental variations.
  5. Spin down all the tubes briefly on a benchtop microcentrifuge and place the tubes on a magnetic rack for 1 min. Remove the supernatant while on the magnetic rack.
    NOTE: It is critical to wait for 1 min after placing the sample tubes on the magnetic rack every time. This can minimize bead loss due to the invisible small amount of beads still moving toward the magnet in the solution.
  6. Wash the beads 2x with 1 mL of Wash Buffer A at RT (5 min rotation each time). Repeat the process with each wash buffer 2x sequentially at 4 °C. (Buffer B, Buffer C, and Buffer D; Table 1).
    NOTE: A high concentration of SDS solution can precipitate at cold temperatures. The first wash must be performed at RT.
  7. Put the tubes on the magnetic rack. Wash the beads 2x with Buffer D to completely remove residual detergent. Resuspend the beads in 100 µL of 50 mM Tris buffer and add 5 mM TCEP (final concentration) to incubate for 30 min at 37 °C in a temperature-controlled mixer, shaking at 1,200 rpm.
  8. Add 15 mM iodoacetamide (IAA, final concentration) to each tube and incubate for 30 min at 37 °C in a temperature-controlled mixer, shaking at 1,200 rpm in the dark (mixer cap on).
    NOTE: IAA is light-sensitive and should be made fresh 5 min before this step.
  9. Add 5 mM TCEP (final concentration) to quench excess IAA. Incubate for 10 min at 37 °C in a temperature-controlled mixer with shaking at 1,200 rpm (mixer cap off).
  10. Centrifuge the sample tubes briefly and place on a magnetic rack for 1 min to remove the supernatant. Add 200 µL of 5 mM TCEP in 50 mM Tris buffer to resuspend the beads. Add 1 µg of Trypsin/Lys-C mix to the sample, and incubate for 14 h at 37 °C in a temperature-controlled mixer, shaking at 1,200 rpm.
  11. Add an additional 0.2 µg of Trypsin/Lys-C mix and digest for 3 h.
  12. Spin down the sample tubes briefly and put the tubes on a magnetic rack for 1 min. Transfer the peptide supernatant to clean the tubes while on a magnetic rack. Wash the beads with 50 µL of 50 mM Tris buffer (shaking for 5 min) and combine the peptide supernatants.
  13. Add 30 µL of 10% trifluoroacetic acid (TFA) to the tube containing peptide supernatant to get pH <3.

6. Peptide desalting and fractionation (2 h)

  1. Wet the reverse-phase solid phase extraction plate (only the wells for use) with 200 µL of HPLC grade methanol (MeOH) 3x on the vacuum manifold. Add 200 µL of 1% TFA in HPLC grade water 3x to equilibrate the plate.
  2. Load the peptide samples into the plate, slowly turning on the vacuum for a flow speed lower than 3 droplets/s to minimize sample loss.
  3. Wash the extraction plate 3x with 200 µL of 1% TFA. Wash the extraction plate again with 200 µL of 1% TFA containing 2% MeOH (v/v). Keep the flow speed lower than 3 droplets/s.
  4. Replace the collection plate with a 96-well collection plate. If no fractionation is needed, elute the peptide samples 3x with 100 µL of 1% TFA containing 80% MeOH and combine in a new sample tube. Dry the peptide samples in a vacuum concentrator with no heat.
    ​NOTE: If fractionation is desired, peptide samples can be eluted into 4 fractions subsequently using 200 µL of 1% TFA containing 15%, 35%, 50%, and 90% MeOH, respectively, in a different collection plate.

7. Colorimetric peptide quantification assay (optional) (1 h)

  1. Resuspend peptide samples in 50 µL of LC-MS grade water and take 20 µL aliquots to perform the peptide assay.
    NOTE: This step consumes a large amount of peptide sample but can provide an accurate quantification of peptide concentration before LC-MS analysis. This is typically conducted only once per sample type for testing before large-scale sample preparation.
  2. Prepare serial dilutions of the peptide standard solutions (provided in the colorimetric assay kit) in the LC-MS grade water. Prepare the working reagent by mixing 50% Reagent A, 48% Reagent B, and 2% Reagent C.
  3. Transfer 20 µL of each peptide standard solution (three replicates) and the unknown peptide sample into a 96-well microplate. Add 180 µL of the working reagent to each well, mix well, and incubate the plate for 30 min at RT.
  4. Read the absorbance at 480 nm in a microplate reader and quantify the concentrations of peptide samples based on the peptide standard curve.

8. LC-MS analysis

  1. Resuspend the peptide samples in LC Buffer A (2% acetonitrile and 0.1% formic acid, LC-MS grade). Centrifuge at 16,500 × g for 10 min at 4 °C to get rid of any possible particulates.
  2. Transfer the supernatant to LC-MS vials. Analyze the samples using a nanoLC-MS instrument.
    NOTE: Detailed LC-MS/MS parameters are instrument-dependent and have been described previously22,25,26.
  3. Generate a custom LC-MS exclusion list with a range of retention times for highly abundant contaminant peptide peaks such as streptavidin and trypsin with 5 ppm mass accuracy22 (e.g., common streptavidin peptide peaks are m/z 402.5435 [charge 3], m/z 603.3117 [charge 2], m/z 654.9733 [charge 3], m/z 678.6812 [charge 3], m/z 1017.5182 [charge 2], etc.; common trypsin peptide peaks are m/z 421.7584 [charge 2], m/z 523.2855 [charge 2], and m/z 737.7062 [charge 3]).

9. Proteomics data analysis

  1. Analyze the LC-MS raw data with proteomics data analysis software such as Proteome Discoverer, MaxQuant27, or MS-Fragger28. Include two FASTA libraries for data analysis: 1) a Swissprot Homo Sapiens reference database; 2) a newly generated universal contaminant FASTA library (https://github.com/HaoGroup-ProtContLib), which was proven to improve proteomics identification and decrease false discoveries29.
  2. Set up the proteomics data analysis parameters with a 1% false discovery rate (FDR) cutoff for protein and peptide spectral matching (PSM) identifications. Select trypsin digestion with a maximum of three missed cleavages, a fixed modification of cysteine carbamidomethylation, and a variable modification of methionine oxidation and protein N-terminal acetylation. Use peptide MS1 peak intensities for label-free quantification. Normalize the peptide intensities to the endogenously biotinylated carboxylase, propionyl-CoA carboxylase (PCCA), to reduce proximity labeling variations, as described previously22.
    NOTE: PCCA normalization can be selected in Proteome Discoverer software by including a PCCA protein sequence FASTA file. Alternatively, PCCA normalization can be conducted in the downstream data analysis.
  3. Export protein-level results from the proteomics software. Remove contaminant proteins prior to statistical analysis29. Remove proteins with only 1 PSM or no quantification result. Conduct protein gene ontology (GO)-term analysis with Enrichr30 and protein network analysis with STRING31.

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Results

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 di...

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Discussion

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 presen...

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Disclosures

The authors declare no competing financial interests.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10% (w/v) Saponin solutionAcros Organics419231000Flourescent Microscopy
AccutaseLife TechnologiesA1110501cell detachment solution, Cell Culture
B27 SupplementFisher Scientific17504044Cell Culture, Cortical Neuron Medium
BDNFPeproTech450-02Cell Culture, Cortical Neuron Medium
Boric acidSigma-AldrichB6768Cell Culture, Borate Buffer
Bovine Serum AlbuminMillipore SigmaA8806To make standard solutions to measure total protein concentrations
Brainphys neuronal mediumSTEMCELL Technologies5790Cell Culture, Cortical Neuron Medium
CD45R (B220) Antibody Alexa Fluor 561Thermo Fisher Scientific505-0452-82Flourescent Microscopy
Chroman1 ROCK inhibitorTocris716310Cell Culture
cOmplete mini Protease InhibitorRoche4693123001cocktail inhibitor in Lysis Buffer
DC Protein Assay Kit IIBio-Rad5000112To determine total protein concentrations of cell lysate
Dimethyl sulfoxide (DMSO)Sigma-AldrichD8418Proximity-labeling Reaction
DMEM/F12 mediumThermo Fisher Scientific11320082Cell Culture, Dish Coating
DMEM/F12 medium with HEPESThermo Fisher Scientific11330057Cell Culture, Induction Medium
Donkey serumSigma-AldrichD9663Flourescent Microscopy
Doxycycline hyclate, ≥98% (HPLC)Sigma-AldrichD9891-1GCell Culture, Induction Medium
Essential 8 MediumThermo Fisher ScientificA1517001Cell Culture
Essential 8 Supplement (50x)Thermo Fisher ScientificA1517101Cell Culture
Extraction plate vacuum manifold kitWatersWAT097944For Peptide desalting
Formic Acid (FA)Fisher ScientificA11750For LC-MS analysis
GDNFPeproTech450-10Cell Culture, Cortical Neuron Medium
Hoechst dyeThermo Fisher Scientific62239Flourescent Microscopy
HPLC grade methanolFisher ScientificA452For Peptide desalting
HPLC grade waterFisher ScientificW5For Peptide desalting
Human induced pluripotent stem cellsCorriell InstituteGM25256Cell Culture
Hydrogen peroxide, ACS, 29-32% w/w aq. soln., stab.Thermo Fisher ScientificAA33323ADProximity-labeling Reaction
Iodoacetamide (IAA)Millipore SigmaI6125For Protein Digestion
LamininFisher Scientific23017015Cell Culture, Cortical Neuron Medium
LC-MS grade AcetonitrileFisher ScientificA955For LC-MS analysis
LC-MS grade waterFisher ScientificW64For LC-MS analysis
L-glutamineFisher Scientific25-030-081Cell Culture, Induction Medium
MatrigelThermo Fisher Scientific08-774-552basement membrane matrix, Cell Culture, Dish Coating
Mouse anti-human LAMP1 monoclonal antibodyDevelopmental Studies Hybridoma Bankh4a3Flourescent Microscopy
N-2 Supplement (100x)Fisher Scientific17-502-048Cell Culture, Induction Medium
Nitrocellulose Membrane, Precut, 0.45 µm, 7 x 8.5 cmBio-Rad1620145To conduct dot blot assay for bead titration
Non-essential amino acids (NEAA)Fisher Scientific11-140-050Cell Culture, Induction Medium
NT-3PeproTech450-03Cell Culture, Cortical Neuron Medium
Oasis HLB 96-well solid phase extraction plateWaters186000309For Peptide desalting
Odyssey Blocking Buffer (TBS)LI-COR Biosciences927-50000To conduct dot blot assay for bead titration
ParaformaldehydeElectron Microscopy Sciences15710Flourescent Microscopy
Phenol Biotin (1,000x stock)Adipogen41994-02-9Proximity-labeling Reaction
Phosphate-buffered saline (PBS) without calcium or magnesiumGibco10010049Cell Culture, Proximity-labeling Reaction, Flourescent Microscopy
Pierce Quantitative Colorimetric Peptide AssayThermo Fisher23275Peptide Concentration Assay
Poly-L-Ornithine (PLO)Millipore SigmaP3655Cell Culture, Dish Coating
Sodium AscorbateSigma-AldrichA4034Proximity-Labeling Quench Buffer, Lysis Buffer
Sodium azideSigma-AldrichS8032Proximity-Labeling Quench Buffer, Lysis Buffer, Flourescent Microscopy
Sodium chlorideThermo Fisher ScientificS271500Cell Culture, Borate Buffer
Sodium dodecyl sulfate (SDS)Thermo Fisher ScientificBP1311220Lysis Buffer, Dot blot assay buffer, Beads wash buffer
Sodium hydroxideSigma-Aldrich415413Cell Culture, Borate Buffer
Sodium tetraborateSigma-Aldrich221732Cell Culture, Borate Buffer
SpeedVac concentratorvacuum concentrator
Streptavidin Magnetic Sepharose BeadsCytiva (formal GE)28-9857-99Enrich biotinylated proteins
Streptavidin, Alexa Fluor 680 ConjugateThermo Fisher ScientificS32358To conduct dot blot assay for bead titration
Thermomixertemperature-controlled mixer
Trifluoacetic acid (TFA)Millipore Sigma302031For Peptide desalting
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)Millipore SigmaC4706For Protein Digestion
Tris-HClThermo Fisher ScientificBP152500Lysis Buffer, Dot blot assay buffer, Beads wash buffer
Triton-XThermo Fisher ScientificBP151500Beads wash buffer
TROLOXSigma-Aldrich648471Proximity-Labeling Quench Buffer, Lysis Buffer
Trypsin/Lys-C Mix, Mass Spec GradePromegaV5073For Protein Digestion
TWEEN 20Millipore SigmaP1379Dot blot assay buffer
UreaThermo Fisher ScientificBP169500Beads wash and On-Beads Digestion Buffer
VitronectinSTEMCELL Technologies7180Cell Culture, Dish Coating
Y-27632 ROCK inhibitorSelleckS1049Cell Culture

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Proteomics AnalysisNeuronal LysosomesiPSC-Derived NeuronsLysosomal Membrane ProteinsBiotinylated Protein EnrichmentLC-MS AnalysisFluorescence MicroscopyOn-Beads Digestion
Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics
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