Apr 28, 2026
Version 2
Liquid Chromatography-Mass Spectrometry (LC-MS) with striatal synaptosomes V.2
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen, Loukia Parisiadou 2026. Liquid Chromatography-Mass Spectrometry (LC-MS) with striatal synaptosomes. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyqk6mvx9/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 28, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 315882
Keywords: ASAPCRN, LC-MS, synaptosome, studies on striatal synaptosome, striatal synaptosome, pathogenic hyperactive lrrk2g2019s mutation exhibit deficit, exhibit deficits in dopamine release, pathogenic hyperactive lrrk2g2019, dopamine release, dopamine, mass spectrometry, synaptosome, quantitative phosphoproteomic liquid chromatography, striatum, underlying molecular mechanism, mice
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
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 quantitative phosphoproteomic Liquid Chromatography-Mass Spectrometry (LC-MS) studies on striatal synaptosomes from Lrrk2G2019S mice.
Safety warnings
These protocols need prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee
LC-MS sample preparation
Mice were administered a dose of MLi-2 (10 mg/kg, 3hours) or control vehicle via oral gavage.
3 hours after administration, striata were dissected and tissues were flash frozen and stored at −80°C until use
Three mouse striata were pooled and rapidly homogenized in four volumes of ice-cold Buffer A supplemented with a Halt protease and phosphatase inhibitor cocktail (Thermo) using a Teflon homogenizer with 12 strokes.
Buffer A recipe (0.32 M sucrose, 5 mM HEPES, pH 7.4, 1 mM MgCl2, 0.5 mM CaCl2)
Centrifuged at 1400 g, 4°C, 10 minutes.
The supernatant (S1) was saved, and the pellet (P1) was homogenized in Buffer A (500ul) with a Teflon homogenizer (5 strokes).
Centrifuged at 700 g, 4°C, 10 minutes.
The supernatant (S1’) was pooled with S1.
Centrifuged at 13,800 g, 4°C, 10 minutes, resulting in a crude synaptosomal pellet (P2) and its corresponding supernatant (S2).
The P2 pellet was resuspended in Buffer B (2.5 ml) supplemented with the protease and phosphatase inhibitor cocktail, using a Teflon homogenizer (5 strokes).
Buffer B recipe (0.32 M sucrose, 6 mM Tris, pH 8.0),
Prepare discontinuous sucrose gradient (0.8 M/1 M/1.2 M sucrose solution in 6 mM Tris, pH 8.0, 3 ml each)
P2 was then carefully loaded onto a discontinuous sucrose
Centrifuged in swinging bucket rotor at 82,500 g, 4°C, 2 hours
Collect synaptic plasma membrane fraction (SPM) located at the interphase between the 1 M and 1.2 M sucrose fractions using a syringe and transfer to clean ultracentrifuge tubes.
Centrifuged at 200,000 g for 30 minutes.
The supernatant was removed and discarded, while the SPM pellet was flash frozen and stored at −80°C.
LC-MS -The SPM pellet samples (processed by Tymora Analytical Operations in West Lafayette, Indianapolis)
200 µL of phase-transfer surfactant lysis buffer (PTS), supplemented with phosphatase inhibitor cocktail 3 (Millipore-Sigma) was added to each tissue sample.
phase-transfer surfactant lysis buffer (PTS) : 12 mM sodium deoxycholate, 12 mM sodium lauroyl sarcosinate, 10 mM TCEP, and 40 mM CAA,
Incubated for 10 minutes at 95°C, pulse-sonicated several times, and then incubated for an additional 5 minutes at 95°C.
Centrifuged at 16,000 × g for 10 minutes, collect the supernatant
Dilute samples with 50 mM triethylammonium bicarbonate (five-fold)
determine protein concentration with BCA assay
The samples were then normalized to 300 ug protein in each
digested with 6 mg Lys-C (Wako) for 3 h at 37°C
6 ug trypsin was added for overnight digestion at 37°C.
The supernatants were collected and acidified with trifluoroacetic acid (TFA) to a final concentration of 1% TFA.
Ethyl acetate solution was added at 1:1 ratio to the samples.
vortexed for 2 min and then centrifuged at 16,000 × g for 2 min
collect the aqueous phase (bottom layer), dried completely in a vacuum centrifuge
desalted using Top-Tip C18 tips (Glygen)
Completely dry the samples in a vacuum centrifuge
phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment kit (Tymora Analytical)
Dry the eluted phosphopeptides in a vacuum centrifuge.
The full phosphopeptide sample was dissolved in 10.5 μl of 0.05% trifluoroacetic acid with 3% (vol/vol) acetonitrile and 10 μl of each sample
injected samples into an Ultimate 3000 nano UHPLC system (Thermo Fisher Scientific).
Peptides were captured on a 2-cm Acclaim PepMap trap column and separated on a 50-cm column packed with ReproSil Saphir 1.8 μm C18 beads. The mobile phase buffer consisted of 0.1% formic acid in ultrapure water (buffer A) with an eluting buffer of 0.1% formic acid in 80% (vol/vol) acetonitrile (buffer B) run with a linear 90-min gradient of 6–30% buffer B at flow rate of 300 nL/min. The UHPLC was coupled online with a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific).
The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 375 to 1,500 with the resolution of 60,000) was followed by MS/MS of the 15 most intense ions (30,000
resolution; normalized collision energy - 28%; automatic gain control target (AGC) - 2E4, maximum injection time - 200 ms; 60sec exclusion].
MS Data analysis
The raw files were searched directly against the mouse database with no redundant entries, using Byonic(Protein Metrics) and Sequest search engines loaded into Proteome Discoverer 2.3 software (Thermo Fisher Scientific).
The data from the two search engines was combined.
MS1 precursor mass tolerance was set at 10 ppm, and MS2 tolerance was set at 20 ppm. Search
criteria included a static carbamidomethylation of cysteines (+57.0214 Da), and variable modifications of phosphorylation of S, T and Y residues (+79.996 Da), oxidation (+15.9949 Da) on methionine residues and acetylation (+42.011 Da) at N terminus of proteins.
Search was performed with full trypsin/P digestion and allowed a maximum of two missed cleavages on the peptides analyzed from the sequence database. The false-discovery rates of proteins and peptides were set at 0.01.
All protein and peptide identifications were grouped and any redundant entries were removed. Only unique peptides and unique master proteins were reported.
All data were quantified using the label-free quantitation node of Precursor Ions Quantifier through the Proteome Discoverer v2.3 (Thermo Fisher Scientific).
For the quantification of phosphoproteomic data, the intensities of phosphopeptides were extracted with initial precursor mass tolerance set at 10 ppm, minimum number of isotope peaks as 2, maximum ΔRT of
isotope pattern multiplets – 0.2 min, PSM confidence FDR of 0.01, with hypothesis test of ANOVA, maximum RT shift of 5 min, pairwise ratio-based ratio calculation, and 100 as the maximum allowed fold change.
For calculations of fold-change between the groups of proteins, total phosphoprotein abundance values were added together and the ratios of these sums were used to compare proteins within different samples.
Threshold forsignificance was set to p<0.05 (unadjusted p-value) & FC (difference on the log2 intensity values) ≥ 0.58.