Apr 15, 2026

in vivo TurboID

Forked from a private protocol
DOI
https://dx.doi.org/10.17504/protocols.io.14egn4b9qv5d/v1
  • Gabi Sejourne1
  • 1Duke University
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn4b9qv5d/v1
Protocol Citation: Gabi Sejourne 2026. in vivo TurboID. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn4b9qv5d/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 19, 2025
Last Modified: April 15, 2026
Protocol  Integer ID: 124627
Keywords: msto map astrocytic endolysosomal protein network, astrocytic endolysosomal protein network, astrocyte, specific in vivo proximity biotinylation, vivo proximity biotinylation, selective biotinylation of protein, subcellular proteome, fusion protein, selective biotinylation, streptavidin affinity capture, protein, using streptavidin affinity capture, labeled protein, biotin administration, protein interaction
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative Grant
Grant ID: ASAP-020607
Abstract
This protocol describes an astrocyte-specific in vivo proximity biotinylation (iBioID) approach using a Lamp1-TurboID fusion protein to investigate protein interactions within the endo-/lysosomal compartment of astrocytes. By leveraging astrocyte-specific AAV delivery in neonatal (P1) mice, this method enables selective biotinylation of proteins in a physiologically relevant context while preserving interactions with neighboring cells. Following biotin administration, labeled proteins are purified using streptavidin affinity capture and analyzed via quantitative LC-MS/MSto map astrocytic endolysosomal protein networks. This approach provides insights into subcellular proteomes and dynamic astrocyte signaling in vivo.
Materials
  • NaCl (Sigma S9888-500G)
  • Tris (Proteomics grade) (VWR 97063-890)
  • SDS (Proteomics grade) (VWR 97064-858)
  • Beckman Coulter Microfuge Tube (Beckman Coulter 357448)
  • Lithium chloride (Sigma L4408)
  • Keratin-free H2O (VWR LC365-1)
  • Low-binding eppendorf tubes (VWR 80077-230)
  • Ammonium bicarbonate (Sigma A6141-500G)
  • Deoxycholate Sigma  (30970-25G)
  • Biotin   Sigma  (B4501-1G)
  • EDTA     Sigma  (4010-250GM)
  • beta-mercaptoethanol (VWR 97064-880)
  • Glycerol (VWR 97063-892)
  • Weigh paper (VWR 12578-121)
  • Spatulas (VWR 80081-190)
  • 30G needles (VWR BD305106)
  • Dounce homegenizer (ceramic) (Sigma P7859)
  • Streptavidin-coated beads (Thermo-Fisher 29202)
  • Triton-X100 (Roche/Sigma 11332481001)
  • cOmplete EDTA-freeProtease inhibitor (Roche/Sigma 4693132001)
TurboID Plasmids
pZac2.1-GfaABC1D-Lck-GCaMP6f was a gift from Dr. Baljit Khakh (Addgene plasmid #52924). pcDNA3-V5-TurboID-NES was a gift from A. Ting (Stanford) (Addgene plasmid #107169).
TurboID was amplified by PCR from the pcDNA3-V5-TurboID-NES plasmid and subcloned into pZac2.1-GfaABC1D vector with an upstream nuclear exit sequence (NES) to generate pZac2.1-GfaABC1D-NES-TurboID-HA.
Lamp1 was subcloned and inserted upstream of TurboID to generate pZac2.1-GfaABC1D-Lamp1-TurboID-HA.
Adeno-associated Virus (AAV) Production
Purified AAVs were produced as described previously (Uezu et al., 2016). Briefly, HEK293T cells were transfected with pAd-DELTA F6, serotype plasmid AAV PHP.eB, and AAV plasmid (pZac2.1-GfaABC1D-Lamp1-TurboID-HA or pZac2.1-GfaABC1D-NES-TurboID-HA).
Three days after transfection, cells were collected in 15 mM NaCl, 5 mM Tris-HCl, pH 8.5, and lysed with repeat freeze-thaw cycles followed by treatment with Benzonase (Novagen 70664) at 37°C for 30 minutes.
Lysed cells were pelleted by centrifugation, and the supernatant, containing AAVs, was applied to an Optiprep density gradient (Sigma D1556, 15%, 25%, 40%, and 60%) and centrifuged at 67,000 rpm using a Beckman Ti-70 rotor for 1 hour.
The AAV-enriched fraction was isolated from between 40% and 60% iodixanol solution and concentrated by repeated washes with sterile PBS in an Amicon Ultra-15 filtration unit (NMWL: 100 kDa, Millipore UFC910008) to a final volume of ∼100 μl and aliquoted for storage at −80°C.
Animal husbandry, AAV administration, euthanasia and brain collection
Order timed pregnant dam from Charles River. Inject half of all pups with AAVs carrying Astro-Lamp-TurboID (PHP.eB.GfaABC1D-Lamp1-TurboID-HA) and half with Astro-CYTO-BioID (PHP.eB.GfaABC1D-NES-TurboID-HA). WT CD1 P1 mouse pups were anesthetized by hypothermia (10 minutes on ice), and inject 1µl of each concentrated AAV-TurboID vector bilaterally into the cortex using a Hamilton syringe. Monitor pups until they recover on a heating pad.
At P18, P19, and P20, inject biotin subcutaneously at 24 mg/kg to increase the biotinylation efficiency.
At P21, anesthetize mice with 200 mg/kg Tribromoethanol (Avertin) and transcardially perfuse with TBS. Dissect the cerebral cortices and flash freeze in liquid nitrogen, and storee at -80°C.
Protein purification
Prepare Buffers in Keratin-free conditions (in hood, using keratin-free H2O)
Prepare stock solutions:
  • 1M NaCl: 2.922g in 50ml keratin-free H2O (KF-H2O)
  • 1M Tris-HCl pH 7.5: 6.057g Tris + ~3.2mL of 12N HCl in 50mL KF-H2O
  • 2M Tris-HCl pH 6.8: 6.057g Tris + ~3.8mL of 12N HCl in 25mL KF-H2O
  • 1M Ambic: 3.95g in 50ml KF-H2O
  • 10% SDS: 5g SDS in 45ml KF-H2O (dissolve with heat)
  • 0.5M EDTA: 9.3g EDTA + 1g NaOH in 50ml KF-H2O
  • 50x Protease Inhibitor in Lysis-R (1 tablet in 500ul, store at -20)
  • 50x Protease Inhibitor in 2x RIPA (1 tablet in 500ul, store at -20)
Prepare Lysis-R Buffer [50mM Tris/HCl pH7.5, 150mM NaCl, 1mM EDTA + Protease inhibitors]:
For 50mL:
  • 2.5mL 1M Tris/HCl pH7.5
  • 7.5mL 1M NaCl
  • 100ul 0.5M EDTA
  • 39.9mL KF H2O
  • Add protease inhibitors as needed (50x)
Prepare 2x RIPA Buffer [50mM Tris/HCl pH7.5, 150mM NaCl, 1mM EDTA, 0.4% SDS, 2%TritonX100, 2% deoxycholate]:
For 50mL:
  • 2.5mL 1M Tris/HCl pH7.5
  • 7.5mL 1M NaCl, 100ul 0.5M EDTA
  • 10mL 10% TritonX100
  • 2mL 10% SDS
  • 1g deoxycholate
Prepare Wash Buffer [1% TritonX100, 1% deoxycholate, 25mM LiCl]:
For 50mL:
  • 5mL 10% TritonX100
  • 0.5g deoxycholate
  • 53mg LiCl
Prepare 50mM Ambic:
For 50mL:
  • 2.5mL 1M Ambic
  • 47.5mL KF H2O
Prepare 2X Sample Buffer in MS-grade water (20mL) [4% SDS, 20% glycerol, 0.1% beta-mercaptoethanol, 125mM Tris pH 6.8 to 20mL with keratin-free water]:
8mL 10% SDS
4mL Glycerol (proteomics grade)
20uL beta-mercaptoethanol
1.25mL 2M Tris pH 6.8
4.75mL KF-H2O
Prepare 2X Sample Buffer in MS-grade water with fresh 5mM biotin:
*ON day of elution
Thaw 1ml aliquot of 2X Sample Buffer in MS-grade water and freshly add 1.2mg biotin. Incubate on rotator for at least 20 minutes to dissolve biotin.
Purify protein and begin incubation with streptavidin beads.
Prepare Lysis-R buffer + protease inhibitors (prepare 1mL per brain). Cool Lysis-R buffer and dounce homogenizers on wet ice. Rinse douncers with diH2O
Take samples from -80 and keep on dry ice.
For each brain, homogenize with 1mL of Lysis-R buffer. Use dounce homogenizer with ceramic plunger. 20 strokes. Homogenize one brain at time.
Transfer homogenate to a 1.5mL epitube (I split the volume between two tubes to make sonication and centrifugation steps easier)
Add 1ml of 2x RIPA buffer (again, split between the two tubes. Now you will have two tubes per brain, each containing approximately 1ml)
Sonicate in BAC core on 4th floor, Room 416E (also reserve ultracentrifuge in Erickson lab).
  • Keep samples on ice at all times.
  • Yellow tape needle is one to use for eppendorf tubes: wash with MilliQ H2O and then clean with Kim wipe.
  • Set to level 9.
  • Submerge needle to the bottom of the tube and turn on for 10 sec.
  • Repeat two more times, 10 sec each.
Centrifuge at 15,000 rpm for 10 min at 4*C
Transfer the supernatant to a Beckman Coulter 5mL (cat# 357448)
Centrifuge in the ultracentrifuge (TLA-55, 40,000 rpm [100,000 g], 30 min, 4*C)
*I have been having difficulty with centrifuge maintaining 40,000rpm, and have been doing the spin at 38,000)
*There is another ultracentrifuge in Chris Nicchita’s lab that worked but I still had to centrifuge at 38,000. Make sure the vacuum has had sufficient time to remove the air before starting (even if the vacuum light is off)
Transfer supernatant to a new Eppendorf Low-protein binding tube and add SDS to a final 1% concentration (sup. Volume x 0.088 of 10% SDS solution)
Brief vortex and spin at room temp
Heat samples (45*C for 45 min)
Cool on ice briefly
Centrifuge 15,000 rpm, 30 min, 4*C
During centrifugation, prepare Neutravidin beads by washing 70ul of slurry (35ug of beads) with 1mL of 2x RIPA and spinning at 5,000 rpm. Repeat for a total of two washes. After the final wash, resuspend in 2X RIPA.
*70ul is the amount to use for one condition, no matter how many brains.
*Note: always prepare more slurry than you will need. For two conditions, I would prepare ~250ul.
Transfer sample supernatant to a 15 mL tube. Pool brains here if applicable (for p21 samples, pool sets of 2 sex- and construct-matched cortices).
*At this step, you can take an aliquot for western blot (30ul from each condition)
Add Neutravidin beads and incubate overnight at 4deg with rotation.
Wash beads and elute protein.
Spin down tubes, 5000rpm for 1min and save supernatant at -80
Transfer beads to a low-binding 1.5mL tube
Wash with 1mL of 2%SDS x2 (10min each). All washes done with rotator or nutator at room temp. Spin down for 2min using the small benchtop (“close & start”) centrifuge. Carefully remove wash solution with P1000. Repeat this process after each wash.
Wash with Wash Buffer (1% TritonX100, 1% deoxycholate, 25 mM LiCl) x2 (10 min each)
Wash with 1 M NaCL x 2 (10 min each) at RT
Wash with 50 mM Ambic x 5 (10 min each) @ RT
During the Ambic washes, thaw aliquot of 2x elution buffer and add 1.2mg of fresh biotin. Incubate on rotator for about 20min (start preparing during 1st or 2nd ambic wash). Most of the biotin will dissolve, but some will still be visible.
Remove the supernatant (use 30G needle to remove as much as possible) and add 2x elution buffer (same amount as slurry ~70ul)
Heat at 60℃, 15 min
Spin down at 5000rpm for 1min. Use a 30G needle and syringe to transfer biotinylated proteins to a new Eppendorf tube and store -80℃ *At this step, you can take a sample (5ul) of the eluate for western blot
LC/MS/MS Sample Preparation
The Duke Proteomics and Metabolomics Shared Resource (DPMSR) receives samples and stores at -80°C until processing.
Samples are spiked with undigested bovine casein at a total of either 1 or 2 pmol as an internal quality control standard.
Samples are reduced for 15 min at 80°C, alkylated with 20 mM iodoacetamide for 30 min at room temperature, then supplemented with a final concentration of 1.2% phosphoric acid and 588 μL of S-Trap (Protifi) binding buffer (90% MeOH/100mM TEAB) (Supplemental Table XXX).
Proteins are trapped on the S-Trap micro cartridge, digested using 20 ng/μL sequencing grade trypsin (Promega) for 1 hr at 47°C, and eluted using 50 mM TEAB, followed by 0.2% FA, and lastly using 50% ACN/0.2% FA.
All samples are lyophilized to dryness.
Samples are resuspended in 12 μL of 1% TFA/2% acetonitrile with 12.5 fmol/μL of yeast ADH.
A study pool QC (SPQC) was created by combining equal volumes of each sample.
LC-MS/MS
Quantitative LC/MS/MS was performed on 3 μL of each sample, using an MClass UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source.
The sample is first trapped on a Symmetry C18 20 mm × 180 μm trapping column (5 μl/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation is performed using a 1.8 μm Acquity HSS T3 C18 75 μm × 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55°C.
Data collection on the Fusion Lumos mass spectrometer is performed for three different compensation voltages (-40v, -60v, -80v).
Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m/z 200) full MS scan from m/z 375 – 1500 with a target AGC value of 4e5 ions is performed.
MS/MS scans with HCD settings of 30% are acquired in the linear ion trap in “rapid” mode with a target AGC value of 1e4 and max fill time of 35 ms.
The total cycle time for each CV is 0.66s, with total cycle times of 2 sec between like full MS scans.
A 20s dynamic exclusion is employed to increase depth of coverage.
The total analysis cycle time for each sample injection is approximately 2 hours.
Quantitative Data Analysis
Peptide alignment and intensity quantification:
Following UPLC-MS/MS analyses (excluding conditioning runs, but including SPQC samples, Table XXX), data are imported into Proteome Discoverer 3.0 (Thermo Scientific Inc.) and individual LCMS data files are aligned based on the accurate mass and retention time of detected precursor ions (“features”) using Minora Feature Detector algorithm in Proteome Discoverer.
Relative peptide abundance is measured based on peak intensities of selected ion chromatograms of the aligned features across all runs.
The MS/MS data is searched against the SwissProt M. musculus database, a common contaminant/spiked protein database (bovine albumin, bovine casein, yeast ADH, etc.), and an equal number of reversed-sequence “decoys” for false discovery rate determination.
Sequest with INFERYS is utilized to produce fragment ion spectra and to perform the database searches.
Database search parameters included fixed modification on Cys (carbamidomethyl) and variable modification on Met (oxidation).
Search tolerances are 2ppm precursor and 0.8Da product ion with full trypsin enzyme rules.
Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer are used to annotate the data at a maximum 1% protein false discovery rate based on q-value calculations.
Note that peptide homology is addressed using razor rules in which a peptide matched to multiple different proteins is exclusively assigned to the protein has more identified peptides.
Protein homology is addressed by grouping proteins that have the same set of peptides to account for their identification.
A master protein within a group was assigned based on % coverage.
Raw intensity values for each identified peptide are presented in Table XXX.
Filtering and peptide normalization:
Prior to normalization, a filter is applied such that a peptide was removed if it was not measured at least twice across all samples and in at least 50% of the replicates in any one single group.
After that filter, samples are total intensity normalized (total intensity of all peptides for a sample are summed then normalized across all samples).
Imputation:
Next, the following imputation strategy is applied to missing values. If less than half of the values are missing in a biological group, values are imputed with an intensity derived from a normal distribution of all values defined by measured values within the same intensity range (20 bins).
If greater than half values are missing for a peptide in a group and a peptide intensity is > 5e6, then it was concluded that peptide was misaligned and its measured intensity is set to 0.
All remaining missing values are imputed with the lowest 2% of all detected values (Table XXX).
Peptide summation and trimmed-mean normalization:
Peptide intensities were then subjected to a trimmed-mean normalization in which the top and bottom 10 percent of the signals were excluded and the average of the remaining values was used to normalize across all samples.
Lastly, all peptides belonging to the same protein were then summed into a single intensity (Table XXX).
These normalized protein level intensities are what was used for the remainder of the analysis.
Quality control assessment:
To assess technical reproducibility, we calculated the % coefficient of variation (%CV) for each protein across 3 injections of an SPQC pool that were interspersed throughout the study (Table XXX).
The mean %CV of the SPQC pools was 9.2%, which is well within our expected analytical tolerances.
To assess biological and technical variability, %CVs were measured for each protein across the individual groups which averaged 18.9%, which is also within our normal tolerances and indicated a reproducible IP and sample processing.
Statistical analysis:
As an initial statistical analysis, we calculated fold-changes between various sample groups based on the protein expression values and calculated two-tailed heteroscedastic t-test on log2-transformed data for this comparison.
Those fold changes and p-values are presented for all proteins in Table XXX.
False Discovery Rate (FDR) correction was performed using the Benjamini-Hochberg procedure, and these values are reported (“p.adjusted”) in Table XXX.
To optimize for discovery, we opted to use the less stringent parameter of unadjusted p-value for downstream analyses.
Within supplemental tables, we have labeled proteins significantly more abundant (“Up”, fold change >2 and unadjusted p-value <0.05), or less abundant (“Down”, fold change >-1.5 and unadjusted p-value <0.05) in a particular genotype or BioID sample group.
These annotated proteins were used for downstream analyses, including protein interaction networks using Cytoscape (v.3.9.1) and Gene Ontology (GO) enrichment analysis using the ClusterProfiler package for R, with all M. musculus genes as the reference background.
Protocol references
Takano, T., Wallace, J. T., Baldwin, K. T., Purkey, A. M., Uezu, A., Courtland, J. L., Soderblom, E. J., Shimogori, T., Maness, P. F., Eroglu, C., & Soderling, S. H. (2020). Chemico-genetic discovery of astrocytic control of inhibition in vivo. Nature, 588(7837), 296–302. https://doi-org.proxy.lib.duke.edu/10.1038/s41586-020-2926-0.

Uezu, A., Kanak, D. J., Bradshaw, T. W., Soderblom, E. J., Catavero, C. M., Burette, A. C., Weinberg, R. J., & Soderling, S. H. (2016). Identification of an elaborate complex mediating postsynaptic inhibition. Science (New York, N.Y.), 353(6304), 1123–1129. https://doi-org.proxy.lib.duke.edu/10.1126/science.aag0821