Quantitative proteomics study of the neuroprotective effects of B12 on hydrogen peroxide-induced apoptosis in SH-SY5Y cells

B12 belongs to the coumarin class of compounds that have been shown to have various physiological and pharmacological activities including anti-inflammatory, antibacterial, and antioxidant. In the present study, we characterised the neuroprotective effects of B12 against H2O2-induced neuronal cell damage in SH-SY5Y cells. Protein expression profiling in combination with pathway analysis was deployed to investigate the molecular events associated with the neuroprotective effects in human neuronal cells using a label-free quantitative proteomics approach. A total of 22 proteins were significantly differentially expressed in H2O2-damaged cells with or without B12 treatment. Bioinformatics analysis using the Cytoscape platform indicated that poly pyrimidine tract binding protein 1 (PTBP1) was highly associated with the protective effect, and western blotting verified that PTBP1 was up-regulated in H2O2 + B12 treatment group, compared with the H2O2 treated group. PTBP RNAi experiments knocked down PTBP expression, which cancelled out the protective effect of B12 on cell viability. Thus, we infer that B12 neuroprotective activity involves up-regulation of PTBP1 and its associated signalling networks following H2O2-induced apoptosis in SH-SY5Y cells. B12 or related compounds may prove to be useful therapeutic agents for the treatment of neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

measurements 14 , but label-free quantitative proteomics is growing in popularity because it is reliable, versatile, and is a cost-effective alternative to labelled quantitation 15 .
Various compounds have antioxidant activity, including vitamin C, ubiquinone lipoic acid, β -carotene, creatine, melatonin, and curcumin 1 . Sung-Soo Kim et al. 16 found that 3,5-dicaffeoylquinic acid (3,5-diCQA) has neuroprotective properties against neuronal cell death caused by H 2 O 2 -induced oxidative stress that operate by suppressing caspase-3 activation and restoring glutathione (GSH) levels. Similarly, Venuprasad and colleagues 17 showed that a hydroalcoholic extract of holy basil (Ocimum sanctum) ameliorates H 2 O 2 -induced neuronal damage by stimulating the antioxidant defence mechanism, and could potentially be used to treat oxidative stress-mediated neuronal disorders. B12 (4-methyl-7-hydroxy-8-(1-hydroxyethyl) coumarin; CN patent No. 201210042735.1) is a compound belonging to the coumarin class that have been shown to possess anti-inflammatory, antibacterial, and antioxidant activities. In the present study, we characterised the neuroprotective effects of B12 against H 2 O 2 -induced neuronal cell damage in SH-SY5Y cells. Moreover, we used protein expression profiling combined with pathway analysis to investigate the molecular events associated with the protective effects, and gained insight into the underlying mechanisms of oxidative damage and neuroprotection.

Neuroprotective effects of B12 on H 2 O 2 -treated SH-SY5Y cells. Cell proliferation was observed in
SH-SY5Y cells after treatment with H 2 O 2 at a range of doses, and cell number decreased with increasing H 2 O 2 concentration. A concentration of 200 μM H 2 O 2 decreased cell number by 50%, and this was chosen for subsequent experiments on the neuroprotective effects (Fig. 1A). Clear differences in cell survival rate were apparent following treatment with B12, and survival varied with B12 concentration in a dose-dependent manner. The neuroprotective effects of B12 were prevalent at > 2 μM, and a 75% cell survival rate was achieved at 200 μM H 2 O 2 (Fig. 1B). Concentrations of 200 μM H 2 O 2 and 20 μM B12 were subsequently used to analyse protein expression in label-free proteomics experiments.

Protein expression analysis.
A total of 4,969 proteins were identified using the shotgun method, and after applying the label-free algorithm (repeated measurement of at least two peptides of each protein in all six samples), 3,505 proteins were successfully quantified. A logarithmic transformation (base 2) of the LFQ intensity of proteins was pre-performed prior to the identification of differentially expressed proteins (Fig. S2). Statistical analysis with Perseus software was performed to select proteins that were differentially expressed following B12 treatment, using the following criteria: fold change > 2, p-value < 0.01 (using the Student's t-test). As a result, we identified 22 significantly differentially expressed proteins in the H 2 O 2 + B12 treatment group (P < 0.01; shown as B12 in figures) compared with the H 2 O 2 treatment group (shown as Ctrl in figures). Of these, 12 proteins were up-regulated and 10 proteins were down-regulated (Table 1). Information on protein intensity and RSD  Table S1. Hierarchical clustering analysis of the 22 significantly differentially expressed proteins was performed ( Fig. 2A), and scatter plots were constructed to determine the fold change in protein expression (x-axis = log2 Ratio (B12/Ctrl), y-axis = log p-value; Fig. 2B). Additional MS and Venn diagram data, as well as scatter plots, are shown in Figs S3, 4). The Venn diagram (Fig. S3) shows the number of identified proteins in the three replicates, including their overlap in the replicates. Scatter plots of protein intensity from all replicates against the Pearson correlation coefficient (Fig. S4) showed a higher correlation within the same group than between different groups.

Bioinformatics analysis.
To construct a protein-protein interaction network associated with B12 regulation, we matched the 22 significantly differently expressed proteins with regulatory data in six publically available databases (DIP, BIOGRID, HPRD, BIND, MINT and INTACT). The resulting network was visualized with Cytoscape 3.1.1 and included 614 nodes and 712 edges (Fig. 3A). The node degree distribution is shown in Fig. 3B. The cluster search algorithm, which considers highly interconnected dense regions within a network, was used to rank network proteins. Protein poly pyrimidine tract binding protein 1 (PTBP1), which was up-regulated in B12-treated cells, was the highest ranked ( Fig. 3C), indicating a potential role in the neuroprotective effects of B12. Gene ontology analysis was then performed using the BinGO plugin in Cytoscape, and genes influenced by B12 were highly correlated with cellular macromolecule metabolic process, cellular metabolic process, nucleic acid metabolic process and gene expression biological process categories ( Fig. 4; Table S2). PTBP1 was also associated with these biological processes (Table S2). The top 10 biological processes are listed in Table S2.
Western blot verification of differentially expressed protein PTBP1. In view of the important role of PTBP1 in the neuroprotective effect of B12, we used western blotting to verify expression in treatment and control groups, and found that PTBP1 was significantly up-regulated in the H 2 O 2 + B12 treatment group (Fig. 5).

PTBP1 knockdown confirmed its involvement in the neuroprotective effect of B12.
After validation of PTBP1 expression by western blotting, we further investigated its role using knockdown experiments. Following treatment with 200 μM H 2 O 2 and 20 μM B12, clear differences were observed in the cell survival rate between the PTBP1 knockdown (siPTBP1) and control (siScramble) groups (Fig. 6A). Specifically, cell survival was increased in the siScramble group but not the siPTBP1 group, and the number of cells was also increased in cells treated with B12 without H 2 O 2 , whereas B12 had no effect on the PTBP1 knockdown group. Knockdown of PTBP1 was confirmed by immunoblotting of transfected cells (Fig. 6B).  3 . In the present study, we used H 2 O 2 as a mediator of oxidative stress in the human neuroblastoma SH-SY5Y cell line, and H 2 O 2 -induced cell damage at a concentration of 200 μM or higher. We discovered that B12 could significantly protect cells from damage induced by H 2 O 2 -induced oxidative stress in a dose-dependent manner. B12 could therefore be used as a drug to protect cells from oxidative damage, and we further investigated the mechanism underlying the neuroprotective effects.
The results of quantitative proteomics and western blotting revealed that PTBP1 protein levels were up-regulated in cells in which B12 was protecting against oxidative stress-induced apoptosis. PTBP1 belongs to a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs), which are RNA-binding proteins that form a complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. Cote et al. 18 observed PTBP1 protein degradation in H 2 O 2 -treated breast cancer cell lines using western blotting. A recent study by King et al. 19 showed that PTBP1 is involved in specific protein sub-complexes that control gene expression during apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL).
PTBP1 knockdown experiments provided more evidence of the important and potentially pivotal role of PTBP1 in the neuroprotective effects of B12, since B12 could no longer rescue cells damaged by oxidative stress after PTBP1 knockdown. Furthermore, the number of cells increased in cells treated with B12 in the absence of H 2 O 2 , indicating an involvement not only in protecting against oxidative stress, but in cells not damaged by H 2 O 2 , although this requires further study. In summary, we can infer that pre-mRNA processing is involved in the neuroprotective effects of B12, and expression of PTBP1, the main target of B12, is required to mediate resistance against H 2 O 2 -induced oxidative damage.
Comprehensive analysis of protein composition, post-translational modifications, and protein dynamics are important goals in cell biology. High performance liquid chromatography mass spectrometry (LC-MS) has established itself as a powerful tool for proteomics studies 20 . Although comprehensive gene expression profiles can be precisely and comprehensively quantified using transcriptomics approaches, transcript levels often show only a modest correlation with the abundance of their corresponding proteins 21 . Moreover, proteins are usually more directly responsible for the regulation of cellular pathways than mRNA transcripts. Thus, protein expression profiling is critical for elucidating the mechanisms underlying the cellular response to specific treatments or drugs 22 . Choi et al. 23 used an LC-MS strategy to separate and identify components of the secretory proteome in adipocytes that are responsive to oxidative stress, and their results indicated that oxidative stress significantly down-regulated MMP-2 secretion and up-regulated TIMP-2 secretion. MS-based proteomic profiling experiments can therefore provide useful and important information to improve our understanding of complex biological processes.
Before analysing the proteomic data to identify significantly differentially expressed proteins in the present study, we first measured the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a house  stress and apoptosis. SANG et al. 24 reported oxidative stress-induced down-regulation of SUMO3 at the transcription level, while Yang et al. 25 observed activation of SUMO2/3 conjugation as a protective response following  brain ischemia/stroke. Our results, together with previous reports, suggest B12 induces the expression of SUMO3 at both mRNA and protein levels, and protects cells from H 2 O 2 -induced oxidative damage, further linking sumoylation to neuroprotection. Mejia et al. 26 reported that Sam68 (KHDRBS1) is recruited into stress granules in response to oxidative stress. In summary, our results provide valuable information on proteins involved in oxidative stress and mediating against oxidative damage.
Interaction networks are useful for studying complex systems in multiple disciplines including economics, sociology, and biology 27 , and can assist the interpretation of the results of proteomics studies. Analysis of proteomics data at the pathway level has become increasingly popular, as demonstrated by Borroto-Escuela et al. 28 who used network analysis to study molecular integration in G protein-coupled receptors (GPCRs) to elucidate the overall architecture of GPCR heteromers. Functional network analysis combined with gene ontology revealed that the vast majority of the significantly differentially expressed proteins identified in the present study were involved in cellular macromolecule metabolic process, cellular metabolic process, nucleic acid metabolic process, cellular component organization and gene expression categories ( Fig. 4; Table S2). It was reported that both triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) were able to activate the coordinated network of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI-3K) pathways 29 . In another study, some of the activated proteins detected functioned in signal transduction pathways associated with the regulation of specific transcription factors 14 . In the present study, the key regulatory protein PTBP1 was associated with cellular macromolecule metabolic process, nucleic acid metabolic process and gene expression biological process categories, consistent with an important role in the neuroprotective effects of B12. Our network analysis and gene ontology results also highlighted other biological processes that are involved in oxidative stress and the anti-oxidative response. Our results provide information on the mechanism underlying the response of cells to H 2 O 2 -induced oxidative stress, and this information may prove useful for the development of treatments for neurodegenerative diseases, although further studies are required to determine the exact mechanism.

Conclusions
The present study indicated that B12 (at concentrations higher than 2 μM) can protect cells from H 2 O 2 -induced cell damage by inducing specific cellular responses at the protein level. Of the differentially expressed proteins, the pre-mRNA processing protein PTBP1 was found to be pivotal. Functional network analysis and gene ontology indicated the neuroprotective effects of B12 are associated with the metabolism of macromolecules and other cellular metabolic processes. Our results provide information on the mechanisms underlying the cellular response to H 2 O 2 -induced oxidative stress, which may prove to be useful for treating chronic neurodegenerative diseases such as Parkinson's and Alzheimer's.
Cell culture and treatment. Cell culture: Human neuroblastoma SH-SY5Y cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin in a humidified atmosphere with 5% CO 2  Analysis of cell viability. SH-SY5Y cells were treated, and cell viability was determined using the conventional MTS reduction assay. Briefly, after treatment, 40 μl of MTS (2 mg/ml in PBS) was added to each well, and cells were incubated at 37 °C for 4 h. Supernatants were aspirated carefully, and the absorbance at 490 nm was measured (in triplicate) with a microplate reader (BIO-RAD Model 3550, CA, USA). Experiments were repeated three times.
Sample preparation for shotgun analysis. In  + B12 treatment group. After determination of the density of the harvested SH-SY5Y cells, aliquots containing 1000 to 10000 cells were collected and placed in individual tubes. Cell pellets were flash frozen in liquid nitrogen and stored at − 80 °C. Proteins were extracted with RIPA lysis buffer, and each group was analyzed in triplicate. A workflow flowchart is shown in Fig. S1. Protein samples (200 μg) from each group were processed according to the manufacturer's protocol for filter-aided sample preparation (FASP) 30 . Proteins were concentrated using Vivacon 500 filtration tubes (Cat No. VNO1HO2, Sartorius Stedim Biotech, UK), mixed with 100 μL of 8 M urea in 0.1 M Tris/HCL (pH 8.5), and centrifuged at 14000 g for 15 min at 20 °C. This step was performed twice, after which 10 μL of 0.05 M Tris-(2-carboxyethyl) phosphine (TCEP) in water was added to the filters, and samples were incubated at 37 °C for 1 h. Then, 10 μL of 0.1 M iodoacetamide (IAA) was added to the filters, after which the samples were incubated in darkness for 30 min. Filters were washed twice with 200 μL of 50 mM NH 4 HCO 3. Finally, 4 μg of trypsin (Promega, Madison, WI) in 100 μL of 50 mM NH 4 HCO 3 was added to each filter. The protein to enzyme ratio was 50:1. Samples were incubated overnight at 37 °C and released peptides were collected by centrifugation.
High pH reverse phase chromatography was performed using the Dionex Ultimate 3000 Micro Binary HPLC Pump system 31 . The digested peptide mixture was reconstituted with 600 μL buffer A (20 mM ammonium formate in water, pH 10) and loaded onto a 2.1 mm × 150 mm Waters BEH130 C-18 column containing 3.5 μm particles. Peptides were eluted at a flow rate of 230 μL/min with a gradient of 5% buffer B (20 mM ammonium formate in 80% acetonitrile, pH 10) for 5 min, 5% to 15% buffer B for 15 min, 15% to 25% buffer B for 10 min, 25% to 55% buffer B for 10 min, and 55% to 95% buffer B for 5 min. The system was then maintained in 95% buffer B for 5 min before equilibrating with 5% buffer B for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 2 min. Fractions containing eluted peptides were pooled into 15 fractions based on peptide density 31 and vacuum-dried for nano-ESI-LC-MS/MS analysis.

LC-MS/MS analysis.
LC-MS experiments were performed on a nano-flow HPLC system (Easy-nLC II, Thermo Fisher Scientific, Waltham, MA, USA) connected to a LTQ-Orbitrap Velos Pro (Linear quadrupole ion trap-Orbitrap mass analyser) mass spectrometer (Thermo Fisher Scientific), equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific). Peptide mixtures (5 μL) were injected at a flow rate of 5 μL/min onto a pre-column (Easy-column C18-A1, 100 μm I.D. × 20 mm, 5 μm, Thermo Fisher Scientific). Chromatographic separation was performed on a reversed phase C18 column (Easy-column C18-A2, 75 μm I.D. × 100 mm, 3 μm, Thermo Fisher Scientific) at a flow rate of 300 nL/min with a 60 min gradient of 2% to 40% acetonitrile in 0.1% formic acid. The electrospray voltage was maintained at 2.2 kV, and the capillary temperature was set at 250 °C. The LTQ-Orbitrap was operated in data-dependent mode to simultaneously measure full scan MS spectra (m/z 350-2000) in the Orbitrap with a mass resolution of 60,000 at m/z 400. After full-scan survey, the 15 most