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.
