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.

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
<ol>
	<li>Human iPSC culture and LAMP1-APEX probe integration (7 days)
	<ol>
		<li>Thaw Matrigel stock solution in an ice bucket at 4 &#176;C ...

<ol>
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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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, &#8805;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 &#181;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&#160;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 di...

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 ...

Lysosomes are the trash disposal systems of the cell. The lysosomal activities are highly dynamic and very difficult to capture. In this study, we developed a proximity labeling proteomics approach to decipher the lysosomal microenvironment in live human neurons.This approach captures the lysosome membrane proteins and the stable and transient interactions with the lysosome and iPSC-derived neurons. Lysosomal dysfunction is heavily implicated in various brain diseases. This technique can be applied to understand various brain diseases and provide potential molecular targets to design new therapies.To begin the procedure for proximity labeling, observe the neurons under a microscope to ensure they are alive and healthy. Take a half-volume of medium from the culture to mix with biotin phenol and add it back to the neurons at a final concentration of 500 micromolar for 30 minutes in a 37 degrees Celsius incubator. Initiate the labeling reaction by adding freshly prepared hydrogen peroxide solution into the neuron culture at one-millimolar final concentration.After a one-minute incubation, aspirate the medium and rinse the culture three times with quench buffer. Tilt the plate to aspirate all the residual buffer. After adding ice-cold lysis buffer, scrape the cell lysates into cold 1.5-milliliter tubes.Sonicate the tubes in an ice-cold water bath at more than 100 watts for 15 minutes with alternate cycles of 40 seconds on and 20 seconds off. After cell lysis, add cold acetone at minus 20 degrees Celsius to the sample at a four-fold volume of the cell-lysate solution. Vortex briefly and incubate at minus 20 degrees Celsius for three hours to precipitate proteins and remove residue biotin phenol.Centrifuge the cell lysate tube at 16, 500 times G for 10 minutes at two degrees Celsius and carefully remove the supernatant without disturbing the protein pellet. Then, wash the pellet twice with one milliliter of acetone at minus 20 degrees Celsius, followed by centrifugation and removal of the supernatant. Next, dry the protein pellet with a vacuum concentrator for one minute.Add cell lysis buffer to completely dissolve the pellet with vortex or sonication. Take 20 microliters of aliquot to determine total protein concentration by the DCA assay. Store the remaining cell solution at minus 80 degrees Celsius.Take the protein lysate sample from minus 80 degrees Celsius freezer and sonicate the sample tube for 30 seconds for quick thawing. Vortex, centrifuge briefly with a benchtop mini centrifuge, and place the sample tube on ice. Next, transfer 250 microliters of streptavidin magnetic bead slurry to each 1.5-milliliter tube.Place these tubes on a magnetic rack for one minute, wash the beads three times with one milliliter of 2%sodium dodecyl sulfate solution, and remove the residual buffer. Based on the cell lysates total protein concentration and the bead titration assay results, add the calculated amount of protein sample needed for 250 microliters of the streptavidin magnetic bead slurry. Then, add cell lysis buffer to the magnetic bead-lysate mixture to the total volume of one microliter and rotate the tube overnight at four degrees Celsius.The next day, centrifuge the sample tube briefly on a benchtop microcentrifuge and place the tube on a magnetic rack for one minute to remove the supernatant. Then, wash the beads twice using one milliliter of wash buffer A with five minutes of rotation at room temperature. Repeat the process with each wash buffer twice, sequentially, with buffer B, buffer C, and buffer D at four degrees Celsius.Place the tube on a magnetic rack for one minute and remove the supernatant. Then, resuspend the beads in 100 microliters of five-millimolar TCEP in 50-millimolar Tris buffer to incubate for 30 minutes on a thermomixer at 37 degrees Celsius, followed by 15-millimolar IAA addition for 30 minutes in the dark and five-millimolar TCEP addition for five minutes to quench residue IAA. Centrifuge the sample tube briefly on a microcentrifuge and place the tube on a magnetic rack for one minute to remove supernatant.Add 200 microliters of five-millimolar TCEP in 50-millimolar Tris buffer to resuspend the beads. Add one microgram of trypsin/Lys-C mix for 14 hours at 1, 200 RPM and 37 degrees Celsius. After 14 hours, add additional 0.2 micrograms of trypsin/Lys-C mix for three hours, spin down the sample tubes, and put them on a magnetic rack for one minute.Then, transfer the peptide supernatant to the clean tubes. Next, wash the beads with 50 microliters of 50-millimolar Tris buffer with shaking for five minutes, combine the peptide supernatants, and add 30 microliters of 10%trifluoroacetic acid to the tube to reduce pH to less than three. After peptide desalting and drying down.resuspend the peptide samples in LC buffer for LC-MS analysis. Transfer the supernatant to the LC sample vial to analyze with nano LC-MS. In the LC-MS method file, Add a custom LC-MS exclusion list with specific masses and retention time ranges for highly abundant contaminant peptide peaks, such as streptavidin, trypsin, and Lys-C.Analyze the LC-MC raw data with proteomics data analysis software. Include two FASTA libraries a Swiss-Prot Homo sapiens reference database, and a newly-built universal contaminant FASTA library with a contaminant prefix in the UniProt ID.Set up the data analysis parameters with a 1%false discovery rate for protein and peptide spectral matching identifications. Select trypsin digestion with three missed cleavages, a fixed modification of cystine carbamidyl methylation, a variable modification of methionine oxidation, and protein end terminal acetylation.For label-free quantification, normalize the peptide MS-I peak intensities to the endogenously biotinylated carboxylase PCCA and reduce the variations in proximity labeling experiments. Export protein level results from the proteomics software as an Excel file. Before the statistical analysis, remove contaminant proteins and proteins with only one PSM or no quantification result.Cell morphologies at different time points of the iPSC neurons illustrated the neurite outgrowth during the three-day differentiation period. After switching to poly-L-ornithine-coated plates in the neuron medium, the neurites formed a network between neurons and axonal extensions became more visible after maturation in two weeks. In the fluorescence imaging, LAMP1 apex activity of biotinylated proteins was stained by streptavidin and co-localized with LAMP1 staining in neurons.The results of the bead titration assay demonstrated a decline of uncaptured biotinylated protein signals when increasing the streptavidin beads amount. Five microliters of streptavidin beads were optimal for 50 micrograms of input proteins from endogenous LAMP1 apex samples. The on-beads digestion with trypsin/Lys-C mix resulted in more protein and peptide identifications and fewer missed cleavages when compared with trypsin alone.Additionally, 1 to 1.5 micrograms of trypsin/Lys-C were found to be optimal for on-beads digestion to obtain the highest number of identified proteins and the lowest percentage of missed cleavages. Known lysosomal membrane proteins were increased in LAMP1 apex versus no apex control. Endogenously biotinylated proteins are highly abundant but remain unchanged.The GO term and protein network analysis suggested that stable lysosomal membrane proteins and transient lysosomal interactors related to endolysosomal trafficking and transport were enriched in LAMP1 apex proteomics. Apex proximity labeling must be quenched at exactly one minute to reduce oxidative stress and experimental variations. We have recently developed a cleavable biotin method that allows us to enrich biotinylated proteins.This method, when combined with our current method, will allow us to reduce the interferences from streptavidin and endogenously biotinylated proteins. We can apply this method to both wild-type and mutant neurons with genetic variants that cause brain diseases. This can help us understand how and why genetic mutations influence lysosomal microenvironment in human neurons.

characterization-neuronal-lysosome-interactome-with-proximity

Research

JoVE Journal

Neuroscience

2.3K vistas.  The George Washington University. Los lisosomas son los sistemas de eliminación de basura de la célula. Las actividades lisosomales son altamente dinámicas y muy difíciles de capturar. En este estudio, desarrollamos un enfoque proteómico de etiquetado de proximidad para descifrar el microambiente lisosomal en neuronas humanas vivas.Este enfoque captura las proteínas de membrana del lisosoma y las interacciones estables y transitorias con el lisosoma y las neuronas derivadas de iPSC. La disfunción lisosomal está...