The SH-SY5Y neuroblastoma cells (Cat # CRL-2266) were grown in a complete culture medium containing 88% of Dulbecco Modified Eagle Medium (DMEM) supplemented with 4.5 g/L glucose and L-glutamine without sodium pyruvate (Corning, Corning, NY, U.S.A), 10% heat-inactivated fetal bovine serum (FBS, Biosera, Nuaille, France), 1% Penicillin-streptomycin (P/S) (Gibco, Carlsbad, U.S.A) and 1% non-essential amino acid (NEAA, GIBCO, Carlsbad, U.S.A) 37oC in a humidified 5% CO2 incubator. The culture medium was replaced every 3-days, and cells were sub-cultured once it reaches 70% confluence.
Differentiation of SH-SY5Y neuroblastoma cells
The establishment of differ-SH-SY5Y neural cells was performed by seeding 1 x 105 cells/mL of SH-SY5Y human neuroblastoma cells in a complete culture medium in a T75 flask. After 24 hours of cell seeding, the cells were exposed to a differentiation medium containing 95% of DMEM, 3% heat-activated FBS, 1% P/S, 1% NEAA and 10 μM of all trans-retinoic acid (RA, Sigma Aldrich, St. Louis, U.S.A) for 6-days. After 6-days of the differentiation phase, the differ-SH-SY5Y neural cells express dopaminergic characteristics at morphological, biochemical and genetic levels (Magalingam et al. 2020).
Preparation and Treatment protocol of 6-hydroxydopamine (6-OHDA)
6-Hydroxydopamine (6-OHDA, Sigma Aldrich, St. Louis, U.S.A) was freshly prepared using chilled 0.15% ascorbic acid (Sigma Aldrich, St. Louis, U.S.A) and sterilized by filtering through a syringe filter fitted with a 0.2 mM filter and stored in the dark at 4oC to protect it from light. On day 7 (after the 6-days of differentiation phase), the differ-SH-SY5Y cells were exposed to 10 μg/mL of 6-OHDA in a serum-free culture medium for 24 hours. The undifferentiated cells were maintained in a serum-free culture medium without 6-OHDA for the same duration of time. The concentration of 6-OHDA (10 μg/mL) was selected based on the preliminary studies that showed that it inhibited proliferation of the differ-SH-SY5Y neural cells by about 50%.
On day 8, the undifferentiated, differ-SH-SY5Y, and 6-OHDA exposed differ-SH-SY5Y neural cells were harvested from the T75 flasks and recovered by centrifugation (1000 g for 10 min at 4oC). The supernatant was discarded, and the total protein was extracted from the cells using the EasyPrep Mini MS Sample Prep kit (Thermo Fisher Scientific, U.S.A). Briefly, 100 µL of lysis buffer (provided with the kit) and 1 µL of universal nuclease (provided with the kit) were added to the cell pellets and thoroughly mixed until the sample's viscosity reduced. The extracted protein samples were aliquoted and stored at -80°C until further use.
Determination of protein concentration
The extracted proteins' concentration was estimated using the Pierce BCA Protein Kit (Thermo Fisher Scientific, U.S.A) (El-Rami et al. 2017). The absorbance was measured at 562 nm using a microplate reader (SpectramaxM, CA, USA). A standard curve was prepared by plotting the average corrected absorbance measurements of BSA standards vs concentration in µg/mL to determine the ‘unknown samples’ protein concentrations.
Protein reduction and alkylation
The extracted proteins were subjected to reduction and alkylation steps followed by protein digestion and a “clean-up” procedure using the EasyPrep Mini MS Sample Prep kit protocol. The samples were removed from the -80°C freezer and thawed at room temperature. Once samples were liquefied, 100 µg of protein from each sample was transferred into appropriately labelled sterile 1.5 mL tubes, and the final volume for each sample was adjusted to 100 µL with lysis solution (provided with the kit). Then, 50 µL of reduction solution (provided with the kit) was added to each tube and mixed gently. Following this, 50 µL of alkylation solution (provided with the kit) was added to the tubes, and the tubes were gently mixed. The tubes were incubated at 95°C using a heat block for 10 minutes to reduce and alkylation reactions. Following this, the samples were removed from the heat block to cool to room temperature and subjected to Trypsin/Lys-C protein digestion procedure.
Trypsin/Lys-C protein digestion
For the digestion step, 500 µL of the enzyme reconstitution solution (provided with the kit) was added to a vial containing Trypsin/Lys-C-Protease mix (provided with the kit). Then, 50 μL of the reconstituted Trypsin/Lys-C-Protease mix was added to each tube containing the samples, and the tubes were incubated at 37°C with shaking for 3 hours to allow protein digestion to take place. At the end of 3 hours, 50 µL of digestion stop solution (provided with the kit) was added to each tube to terminate the digestion process.
After the peptide digestion, any contaminants present in the samples were sequentially removed using the peptide clean-up column (provided with the kit). The peptide clean-up columns were labelled accordingly to avoid any sample mix-ups. As per the manufacturer recommended protocol, the white cap at the bottom of each peptide clean-up column was removed, and its green top cap loosened before each clean-up column was placed in individual 2 mL tubes. The tubes were centrifuged (3000 g for 2 minutes) to remove trapped liquid from the column, and the flow-through from each column was discarded. The digested peptides from each sample were transferred into the respective peptide clean-up column. The columns were centrifuged (1500 g for 2 minutes), and the flow-through from each column was discarded. Next, wash solution A (provided with the kit) was added into each column before centrifugation, and the flow-through from each column was discarded. This step was repeated using wash solution B (provided with the kit). Before the elution step, the peptide columns were centrifuged to remove any residual fluid. Then, each column was placed on appropriately labelled sterile collection tubes, and elution solution (provided with the kit) was added to each column. The eluted peptide samples were collected by centrifugation, dried using a vacuum centrifuge and stored at -80°C before LC-MS/MS analysis.
Liquid Chromatography and Mass Spectrometry Analysis
The digested peptides were loaded into an Agilent 1200 HPLC-Chip/MS Interface, coupled with Agilent 6550 iFunnel Q-TOF LC/MS (Agilent, Santa Clara, CA, USA). The column was equilibrated with 0.1% formic acid in water (solution A). The peptides were eluted from the column with 90% acetonitrile in 0.1% formic acid in water (solution B). Quadrupole-time of flight (Q-TOF) polarity was set at positive with capillary and fragmenter voltage being set at 1900 V and 360 V, respectively, and 5 L/min of gas flow with a temperature of 325 ◦C. The collision energy was determined at 3.7V (100Da), and reference masses with positive polarity was set at 299.294457 and 1221.990637. The peptide spectrum was analysed in auto MS mode ranging from 110–3000 m/z for MS scan and 50–3000 m/z for MS/MS scan.
The raw data of tryptic peptides were extracted and processed using PEAKS X software (Bioinformatics Solutions Inc., Waterloo, ON, Canada) using Uniprot, Swissprot and TrEMBL databases. The PEAKS X software allows for the determination of the protein abundance using the following search parameters: retention time lower bound: ≥ 0, retention time upper bound: ≤ 55, average area: ≥ 0, charge lower bound: ≥ 1, confident number samples per group: ≥ 1, peptide identification count: ≥ 1, protein significance: ≥ 20, used peptides: ≥ 1, fixed modification: Carbamidomethylation of cysteine residues and false discovery rate (FDR): 1% in three biological replicate injections. Protein abundance was computed using normalized spectral protein intensity (LFQ intensity). The obtained peptide/protein list was exported to Microsoft Excel to quantitate three biological replicates from the same samples were grouped in the same matrix. The protein data were filtered for at least two valid values, and protein only presented in one biological replicate was eliminated. The biological replicates from all samples were clustered under the same matrix, and the missing data were assigned with a random number derived from a normal distribution. The reason for the missing data measurement is due to low protein abundance in LC-MS/MS analysis.
Biocomputational Protein analysis
Protein Functional classification was performed using the gene ontology database
The identified proteins with p<0.05 against their respective control were classified based on (i) molecular function, (ii) biological process and (iii) cellular component using Gene Ontology (GO) term analysis. The online bioinformatics tool PANTHER database (http://pantherdb.org) version 16 was used to elucidate the functions of these differentially expressed proteins in the differ-SH-SY5Y neural cells and 6-OHDA exposed differ-SH-SY5Y cells. Only the top 10 enriched GO terms were listed for each functional classification. All results displayed expressed adjusted p-value < 0.05 as determined by Fischer's Exact test and FDR.
Protein-Protein Interaction (PPI) Analysis
The STRING database (STRINGv11.0) (https://string-db.org) was used to construct the PPI network in neuronal maturation and oxidative stress in 6-OHDA treated neural cells based on its physical binding and regulatory interaction. The Uniprot IDs of the differentially regulated proteins were inputted in the STRING database under the multiple protein analysis categories and followed by the selection of Homo Sapiens from organism pull-down selection. The basic settings that were used in the analysis of PPI are Network type: Full network, active interaction sources: text mining, neighbourhood, experiments, databases, co-expression, gene-fusion, co-occurrence, Minimum required interaction score: highest confidence (0.9) and K-mean clustering was specified as three clusters. Thick edges between the protein nodes demonstrate strong protein interaction. This protein cluster was uploaded into Cytoscape 3.8.0 to visualise the complex networks by integrating Log2 fold-change data.
Pathway Enrichment Analysis
The pathway enrichment analysis of the differentially expressed proteins was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery, https://david.ncifcrf.gov/) bioinformatic online database. The cluster with the most enriched proteins exhibiting strong PPI identified from STRING analysis was uploaded as an official gene symbol in DAVID resources. The enriched pathway curated by KEGG (Kyoto Encyclopaedia of Genes and Genomes) was used to elucidate the differentially regulated protein molecular mechanisms.
Statistical analysis comparing the quantitative data from differential protein expression between differ-SH-SY5Y neural cells vs undifferentiated SH-SY5Y cells and 6-OHDA treated differ-SH-SY5Y neural cells vs untreated differ-SH-SY5Y cells were performed using a two-tailed Student's t-test. All statistical analyses were performed with GraphPad Prism version 9.0. Differentially expressed proteins that displayed the difference in Log2 Fold change (< or > 0) with p<0.05 were regarded as statistically significant.