Apr 29, 2026
Version 2
Axonal proteome analysis and western blot via APEX2 labeling V.2
- Chuyu Chen1,
- Yevgenia Kozorovitskiy2,
- Loukia Parisiadou1
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815;
- 2Department of Neurobiology, Northwestern University, Evanston, IL, USA
Protocol Citation: Chuyu Chen, Yevgenia Kozorovitskiy, Loukia Parisiadou 2026. Axonal proteome analysis and western blot via APEX2 labeling . protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjb79bgk5/v2Version created by Chuyu Chen
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: April 29, 2026
Last Modified: April 29, 2026
Protocol Integer ID: 315891
Keywords: ASAPCRN, APEX2 biotinylation , dopamine axon, axonal proteome analysis, exhibit deficits in dopamine release, pathogenic hyperactive lrrk2g2019s mutation exhibit deficit, dopamine release, dopamine, underlying molecular mechanism, vulnerable aldh1a1, pathogenic hyperactive lrrk2g2019, subtle proteomic changes across comparison, aldh1a1, subtle proteomic change, molecular mechanism, axon, western blot via apex2, striatum
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: Grant ID: ASAP-020600
Abstract
We and others previously reported that knock-in mice expressing the pathogenic hyperactive Lrrk2G2019S
mutation, exhibit deficits in dopamine release within the striatum. To investigate the underlying molecular mechanisms, we conducted axonal proteome analysis of a subcluster within vulnerable Aldh1a1+ dopamine axons, defined by Annexin 1 (Anxa1+), confirmed subtle proteomic changes across comparisons.
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APEX2 biotinylation in acute brain slices and tissue dissections
1) AAV5-CAG-DIO-APEX2-NES (Addgene plasmid #79907, virus packaged by VectorBiolabs; titer: 3.8x10¹² GC/ml), delivered bilaterally for APEX2 proximity labeling.
2)The homozygous Cre-dependent APEX2 mouse line was crossed with the DatCre line to achieve expression of APEX2 in dopamine neurons of the substantia nigra compacta (SNc).
Mice were anesthetized with ketamine and underwent transcardial perfusion with 10 ml of ice-cold cutting solution saturated with 95% O2 and 5% CO2.
Cutting solution recipe: 110 mM choline, 2.5 mM KCl, 1.25 mM monosodium phosphate, 10 mM glucose, 7 mM MgCl2, 0.5 mM CaCl2, 1.3 mM NaH2PO4, and 25 mM sodium bicarbonate.
quickly extract the brain and place in ice-cold cutting solution saturated with 95% O2 and 5% CO2.
Prepare coronal slices (300 µm) with Leica VT1200S.
Transfer slices to a glass dish containing artificial cerebrospinal fluid (aCSF), supplemented with 0.5 mM biotin-phenol. The aCSF was continuously saturated with 95% O2 and 5% CO2. incubate for 60mins, RT
artificial cerebrospinal fluid (aCSF) recipe: 2.5 mM KCl, 10 mM glucose, 125.2 mM NaCl, 0.3 mM NaH2PO4, 1.3 mM MgCl2, 2.4 mM CaCl2, 26 mM NaHCO3, and 0.3 mM KH2PO4, supplemented with 0.5 mM biotin-phenol.
Add 1 mM H2O2 to the aCSF, 5 minutes, RT.
The slices were rapidly transferred to a separate glass dish containing quenching aCSF (aCSF supplemented with 10 mM Trolox, 20 mM sodium ascorbate, and 10 mM sodium azide) 5 minutes, RT
Rapidly dissect the slices in ice-cold quenching aCSF. Flash-frozen the collected tissue on dry ice and stored at -80°C until further western blot or proteomic experiments.
5. Protein extraction, and streptavidin enrichment of APEX2 samples
Homogenize frozen APEX2-labeled tissues in 0.75ml ice-cold tissue lysis buffer on ice using a glass Dounce homogenizer with 30 strokes of both A and B pestles.
Tissue lysis buffer recipe: 50 mM Tris (pH 8.0), 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 5 mM Trolox, 10 mM sodium ascorbate, 10 mM sodium azide, and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific).
Add 39 μl of 10% SDS to achieve a final concentration of 0.5%, incubate on a rotary wheel for 15 minutes, 4°C
Centrifuge at 21,000 × g for 10 minutes at 4°C.
Transfer supernatans to a new prechilled Eppendorf tube for trichloroacetic acid (TCA) precipitation (for LC-MS) or stored at −80°C (for western blot).
Add equal volume of ice-cold 55% trichloroacetic acid (TCA) and incubate on ice for 30 minutes
Centrifuge at 21,000 × g for 10 minutes at 4°C.
Resuspend protein pellets in 1 ml of acetone (prechilled to -20°C)
Centrifuge at 21,000 × g for 10 minutes at 4°C.
Repeat steps 15-16, for a total of 4 washes
Remove any residual acetone, the protein pellets were resuspended (500ml) in the Urea Dissolve Buffer
Urea Dissolve Buffer recipe: 8 M urea, 1% sodium dodecyl sulfate (SDS), 100 mM sodium phosphate (pH 8), and 100 mM ammonium bicarbonate (NH4HCO3).
sonication for 1 minute, gentle agitation on an orbital shaker for 1 hour at RT
A small aliquot (5%) of the resuspended protein was flash-frozen and stored at -80°C.
Diluted the rest of the sample (95%) with water to achieve a final concentration of 4 M urea and 0.5% SDS.
Resuspend streptavidin magnetic beads (Thermo Fisher #88817)
wash in Urea Detergent Wash Buffer, 15 mins, 4°C x3
Urea Detergent Wash Buffer : 4 M urea, 0.5% SDS, 100 mM sodium phosphate, pH 8
Resuspend streptavidin magnetic beads in ice-cold Urea Detergent Wash Buffer
Add 0.5 mg/50ml streptavidin magnetic beads (from step 20.2) to each sample from step 18.3
Incubate overnight on a rotary wheel at 4°C for 14-18 hours.
Place the tubes on the magnetic stand for 1 min
Discard unbound supernatant.
Resuspend the beads in 1 ml of Urea Detergent Wash Buffer and transfer to a new tube.
wash in Urea Detergent Wash Buffer, 10 mins, RT x3
Resuspend the beads in 1 ml of Urea Wash Buffer and transfer to a new tube.
Urea Wash Buffer: 4 M urea, 100 mM sodium phosphate, pH 8
wash in Urea Wash Buffer, 10 mins, RT x3
Resuspend the beads in 200 μl of Urea Wash Buffer and transfer to a new tube.
Remove the buffer using a magnetic stand. flash-frozen the beads and stored at -80°C for western blot or LC-MS.
Western blot analysis
Determining protein concentration of frozen tissue lysates using the BCA assay (Thermo Scientific)
10 μg APEX2 labeled tissue lysate (from step 12) was loaded into 4–12% NuPAGE Bis-Tris PAGE (Invitrogen) as input.
Resuspend the streptavidin beads in ~20 μl of 1× SDS sample buffer supplemented with 20 mM DTT and 2 mM biotin.
Boil samples for 5 min at 95°C to elute biotinylated proteins.
Immediately placed the beads onto a magnetic rack and the entire sample was immediately loaded into 4–12% NuPAGE Bis-Tris PAGE (Invitrogen)
Transfer the NuPAGE to membranes using the iBlot nitrocellulose membrane blotting system (Invitrogen)
Incubate the membranes with primary antibodies specific for streptavidin-HRP (1:1000, Abcam), LRRK2/Dardarin clone N137/6 (1:1000, NeuroMab), Th (1:1000, Millipore), Rab3a (1:1000, Sigma), Rab3c (1:1000, Proteintech) overnight at 4°C
Wash membranes with TBS/Tween-20, 10 mins x3
Incubate the membranes with secondary anti-mouse or anti-rabbit antibodies (1:2000, Thermo Scientific) for 1 hour at room temperature.
Wash membranes with TBS/Tween-20, 10 mins x3
Incubate the membranes with Immobilon ECL Ultra Western HRP Substrate (Millipore) for 3 minutes
Chemiluminescent blots were imaged using the iBright imaging system (Thermo Fisher Scientific).
LC-MS (This analysis was performed in Biognosys AG (Schlieren, Switzerland).)
Samples were solubilized and digested overnight with sequencing grade trypsin (Promega) in a urea-containing denaturation buffer. Each sample is the eluted proteins from one mouse
Beads were collected on a magnetic rack, supernatant was transferred to a new tube and used for the clean-up.
Purification for mass spectrometry was carried out using Oasis HLB μElution Plate 30μm plate (WATERS) according to the manufacturer’s instructions.
Peptides were dried down to complete dryness using a SpeedVac system and dissolved in LC solvent A (1 % acetonitrile in water with 0.1 % formic acid (FA)) containing Biognosys’ iRT-peptide mix for retention time calibration.
Peptide concentrations in mass spectrometry ready samples were measured using the mBCA assay (Thermo Scientific Pierce).
For DIA LC-MS/MS measurements, peptides were injected on an in-house packed reversed phase column on a Thermo Scientific EASY-nLC 1200 nano-liquid chromatography system connected to a Thermo Scientific Orbitrap Exploris 480 mass spectrometer equipped with a Nanospray Flex ion source and a FAIMS Pro ion mobility device (Thermo Scientific).
LC solvents were A: water with 0.1 % FA; B: 80 % acetonitrile, 0.1 % FA in water. The nonlinear LC gradient was 1 – 50 % solvent B in 171 minutes followed by a column washing step in 90 % B for 7 minutes, and
a final equilibration step of 1 % B for 0.5 column volumes at 64 °C with a flow rate set to a ramp between 450 to 271 nl/min (min 0: 450 nl/min, min 172: 271 nl/min, washing at 400 nl/min).
MS Data analysis (This analysis was performed in Biognosys AG (Schlieren, Switzerland).)
The DIA mass spectrometric data were analyzed using Spectronaut software (Biognosys,
version 19.0). The false discovery rate on peptide and protein level was set to 1 %.
A mouse UniProt .fasta database (Mus musculus, 2024-07-01) was used for the search engine, allowing for 2 missed cleavages, carbamidomethylation of cysteine as fixed modification and up to 5 variable
modifications (N-terminal acetylation, methionine oxidation, deamidation of asparagine or glutamine and phosphorylation at serine or threonine or tyrosine).
HRM mass spectrometric data were analyzed using Spectronaut software (Biognosys, version 19.0). The false discovery rate on peptide and protein level was set to 1 %, data was filtered using row-based
extraction.
The directDIA+ library generated in this project was used for the analysis. The HRM measurements analyzed with Spectronaut were normalized using global median normalization.
Proteins that were not enriched (enriched: q-value < 0.05, log2fc > 0) in Anxa1Cre; Lrrk2G2019S vehicle
vs. non-transgenic were removed from the dataset of samples from SNc and striatum, respectively. The filtered dataset was used for further analysis.
For testing of differential protein abundance, protein intensities for each protein were analyzed using a two-sample Student’s t-test. The following thresholds were applied for candidate identification: p-value < 0.05; absolute average log2 ratio > 0.58 (fold-change > 1.5).
Distance in heat maps was calculated using the “manhattan” method, the clustering using “ward.D” for both axes. Principal component analysis was conducted in R using prcomp and a modified ggbiplot
function for plotting. Functional analysis was performed using String-db (string-db.org, version 12.0)
Phospho-site summarization was carried out using a PTM localization probability cutoff of > 0.75 and linear model as aggregation type.
For testing of differential phospho-site abundance, phosphosite intensities for each protein were analyzed using a two sample Student’s t test. The following thresholds were applied for candidate identification: p- value <0.05; absolute average log2 ratio > 0.58 (fold change > 1.5).