Quantitative analysis of TMT phosphorylation modification to investigate the effect of verbascoside on the expression of phosphorylated protein in AD cell model


 Ethnopharmacological relevance— The active monomer Verbascum glycosides in Cistanche tubulosa has good development prospects in terms of neuroprotection and delaying neurodegenerative diseases, and it has become one of the research hot spots. Aim of the study— To investigate the effect of verbascoside (OC1) on the expression of phosphorylated protein in the protective effect of AD cell model by TMT labeling and phosphorylation enrichment technique and high-resolution liquid chromatography-mass spectrometry quantitative proteomics research strategy. Materials and Methods— The normal control group, the model group (Aβ 1-42 10μmol/L) group and the OC1 administration group (10μg/ml) group were set. (1)protein extraction quality control.(2)TMT mark.(3)HPLC classification and modification enrichment. (4)Analysis of mass spectrometry by liquid chromatography-mass spectrometry. (5)Analysis of bioinformatics results.(6)Western Blotting was used to detect the expression levels of p-CaMKII(Thr286), p-Synapsin (Ser9) / Synapsin, Synaptophysin and Synaptotagmin-1 protein. Results— The study finally identified 9020 phosphorylation sites on 3227 proteins, of which 8635 sites of 3134 proteins contained quantitative information. Screening of differential sites follows the following criteria: 1.2 times the change threshold and CV value < 0.1. Based on the above data and standards, we performed a systematic bioinformatics analysis of proteins containing quantitative information sites. Western Blotting results showed that Verbascoside could promote the expression of p-CaMKII (Thr286), p-Synapsin (Ser9) / Synapsin, Synaptophysin and Synaptotagmin-1 protein. Conclusions— Verbascoside (OC1) can increase the expression of phosphorylated protein in AD cell model, which provides a basis for further study on the molecular mechanism of verbascoside promoting neurotransmitter release.


Background
Cistanches Herba is a very important TCM that was rst recorded in Shen Nong Ben Cao Jing. Cistanche deserticola is a succulent stem of the genus Cistanche of the genus Cistanche of the genus C. cerevisiae. Chemical analysis of Cistanches Herba revealed that phenylethanoid glycosides, iridoids, lignans, oligosaccharides, and polysaccharides were the main constituents. However, it has been regarded as a representative drug of Chinese medicine in the past (Ma Dongni et al.,2019). Acteoside (verbascoside), one of the main active phenylethanoid glycosides from Cistanche deserticola has demonstrated wide pharmacological activities, such as neuroprotective, immunomodulatory, anti-in ammatory, hepatoprotective and anti-oxidative, etc. (Zhang Qing et al.,2018;Yuan Yating et al., 2016;LIN Huimin et al., 2016). Previous studies have shown that acteoside can markedly reduce cerebral injury in mice induced by D-galactose (Gao et al., 2013), regulate the activity of Acetylcholine transferase(ChAT) and Acetylcholinesterase(AChE) in brain tissue, protects neurons in hippocampal CA1 region of brain tissue, improves brain tissue and immune organs index, and restores brain damage in model mice (Gao Li et Hongyan et al.,2015). Furthermore, verbascoside can signi cantly ameliorated the cognitive dysfunction caused by Aβ 1-42 via blocking amyloid deposition, reversing cholinergic and hippocampal dopaminergic neuronal function (Hu Hang et al., 2016;Shiao YJ et al., 2017;Bai, P et al., 2013). In this way, we concluded that Cistanche tubulosa has a certain application value in the pathogenesis of learning and memory disorders such as Alzheimer's disease(AD).
Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by cognitive de cits. The disease is mainly characterized by memory loss,language impairment, visual spatial skills damage, slow thinking,distracted attention and affective disorder and personality changes, accompanied by the decline of social activity ability and self-life ability (Larson M E et al.,2017). Recent studies support that the neuron toxicity induced by amyloid β peptide (Aβ) (plaques) and protein tau (tangles) aggregation are closely related to AD pathogenesis. (Kenney,K et al., 2018). Insoluble Aβ oligomers aggregate in extracellular plaques and were reported to lead to synaptic dysfunction, neuron toxicity and cell death. The establishment of AD animal model and cell model by the infusing Aβ 1-42 into the lateral ventricle is one of the commonly used tools for studying the pathogenesis and drug treatment of AD(Tian Xinhong et al., 2017).
We have previously found that verbascoside inhibits the apoptosis of neuronal cells induced by Aβ during the pathogenesis of AD, and ameliorate the cognitive dysfunction caused by Aβ 1-42 via blocking amyloid deposition, reversing cholinergic and hippocampal dopaminergic neuronal function. ( Xing Haiyan et al., 2018;Miao Xin et al., 2017;Miao Xin et al., 2017;Ju Bowei et al., 2017). It suggests that verbascoside may be an candidate neurological diseases treatment, which is associated with learning and memory dysfunction. In this study, we screened and validated the phosphorylation protein related to the protective effect of verbascoside on AD cell model, which provided a basis for further study on the molecular mechanism of verbascoside promoting neurotransmitter release.

Experimental methods
Through the organic combination of TMT labeling, high performance liquid chromatography grading technology, phosphorylated peptide modi cation and mass spectrometry-based quantitative proteomics technology, study of Phosphorylation Quantitative Omics in Samples. Its technical route is: Protein extraction-trypsin digestion-TMT/iTRAQ label-a nity enrichment-HPLC fractionation-mass spectrometry-database search-bioinformatics analysis.

Protein extraction
PC12 cells were cultured in high glucose DMEM containing 10% horse serum, 5% fetal bovine serum of penicillin-streptomycin and were maintained in a humidifed atmosphere containing 5% CO2. PC12 cells were divided into Control group, Model Aβ 1-42 (10μmol/L)) group and OC1 (10μg/mL) group, and the cells were scraped gently after 24 hours of culture, then were sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (8M urea, 1% Protease Inhibitor Cocktail). The remaining debris was removed by centrifugation at 12,000 g at 4℃for 10 min. Finally, the supernatant was collected and the protein concentration was determined with BCA kit according to the manufacturer's instructions.

Trypsin digestion and TMT labeling
Dithiothreitol was added to the protein solution to a nal concentration of 5mM and reduced at 56°C for 30min. Iodoacetamide was then added to a nal concentration of 11mM and incubated for 15 min at room temperature in the dark. Finally, the urea concentration of the sample is diluted to less than 2M. Add trypsin and digest at 37 °C overnight. The tryptic digested peptides were desalted and dried in vacuo. The peptide was solubilized with 0.5M TEAB and the peptides were labeled according to the TMT kit instructions.

HPLC classi cation and modi cation enrichment
The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column. Brie y, peptides were rst separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. After incubating the peptide in enrichment buffer, the resin was washed three times. Finally, the modi ed peptide was eluted with 10% ammonia water, and the eluate was collected and vacuum-dried and drained. After draining, removing salt 2.6 Liquid Chromatography-Mass Spectrometry Analysis Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient setting: 0-40 min,4%~22% B;40-52 min, 22%~35% B;52-56 min,35%~80% B;56-60 min,80% B, The ow rate was maintained at 350 nL/min. The peptides were separated by ultra-high performance liquid phase system and injected into the NSI ion source for ionization and then analyzed by Q Exactive Plus mass spectrometry. The resulting MS/MS data were processed using Maxquant search engine 2.7 Mass spectrometry control As shown in the Fig.1, most of the peptides are distributed in 7-20 amino acids, which is consistent with the general rule based on trypsin enzymatic hydrolysis and HCD fragmentation. Peptides with less than 5 amino acids are too small to produce effective sequence identi cation. Peptides larger than 20 amino acids are not suitable for fragmentation of HCD due to their high mass and charge number. The distribution of peptide lengths identi ed by mass spectrometry meets quality control requirements. Gene Ontology analysis: Gene Ontology analysis, or GO analysis, is a bioinformatics analysis method that can link information between genes and proteins to provide statistical information. Gene Ontology analysis mainly includes three aspects: 1. Cell composition 2. Molecular function 3. Biological process.
KEGG pathway annotation: KEGG is able to integrate currently known protein interaction network information. We used the KEGG pathway database to annotate the protein pathway to match the protein to the corresponding pathway in the database. Subcellular localization: Proteins in eukaryotic tissue cells are located in detail on various elements within the cell, depending on the membrane structure with which they bind. We used a software for predicting subcellular localization wolfpsort to perform subcellular localization annotation of the submitted protein.
2.8.2Functional Enrichment 2.8.2.1 Enrichment of Gene Ontology analysis Proteins were classi ed by GO annotation into three categories: biological process, cellular compartment and molecular function.For each category, a two-tailed Fisher's exact test was employed to test the enrichment of the differentially modi ed protein against all identi ed proteins. The GO with a corrected pvalue less than 0.05 is considered signi cantly.

Enrichment of pathway analysis
Encyclopedia of Genes and Genomes(KEGG) database was used to identify enriched pathways by a twotailed Fisher's exact test to test the enrichment of the differentially modi ed protein against all identi ed proteins. The pathway with a corrected p-value less than 0.05 was considered signi cant. These pathways were classi ed into hierarchical categories according to the KEGG website.

Enrichment of protein domain analysis
For each category proteins, InterPro(a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites)database was researched and a two-tailed Fisher's exact test was employed to test the enrichment of the differentially modi ed protein against all identi ed proteins. Protein domains with a corrected p-value less than 0.05 were considered signi cant.

Cluster analysis based on protein function enrichment
Cluster analysis based on functional enrichment of differentially modi ed proteins to study their potential associations and differences in the KEGG pathway. We rst collect functional classi cation information enriched for the protein used and the corresponding enriched P-value values, and then screen out functional classi cations that are signi cantly enriched in at least one protein group. The P-value data matrix obtained by the screening is rst subjected to logarithmic transformation with -log10, and then the transformed data matrix is subjected to Z transformation for each function classi cation. Finally, the data set obtained after Z transformation is analyzed by hierarchical clustering. The clustering relationship is visualized using the heat map drawn by the function heatmap.2 in the R language package gplots.

Analysis of modi ed site motifs
Software MoMo, the motif-x algorithm was used to analyze the motif features of the modi ed sites. Among them, 10 identi ed phosphorylations from upstream and downstream of each identi ed modi cation site were peptide fragments consisting of 6 amino acids from upstream to downstream.The analysis background was 10 upstream and downstream of all potential modi cation sites in the species.
2.9 Western blotting PC12 cells were harvested and washed twice with PBS solution after drug treatment. cells were lysed using RIPA Lysis Buffer. The lysates were collected by scraping from the plates, and then they were centrifuged. Total protein samples were denatured and loaded on a SDS-polyacrylamide gel for electrophoresis, and then transferred onto PVDF transfer membranes. Membranes were blocked at room temperature for 2 h with blocking solution. Membranes were incubated overnight at 4 •C with primary antibodies:p-CaMKII(Thr286)(1:1000),Synapsin(1:2000),p-Synapsin(Ser603)(1:1000), Synaptophysin(1:1000), Synaptotagmin-1(1:1000) and β-Actin(1:1000)dilution in blocking solution. After three washings, the membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G. Then the proteins were detected using an enhanced chemiluminescence detection kit. The images were obtained using a UVP ChemStudio gel imaging system (Analytik Jena AG) and the band intensities were quanti ed with ImageJ software.

Statistics
Results are shown as the mean ± SD. Diferences between two experimental conditions were evaluated with one-way ANOVA. The data were analyzed using SPSS 22.0 software, and a value of p < 0.05 was considered to indicate a statistically signi cant difference.

Overview of modi cation identi cation
In this project, a total of 53460 secondary spectra were obtained by mass spectrometry. After the mass spectrometry secondary spectrum was searched by protein theory data, the available effective spectrum number was 19226, the spectrum utilization rate was 36.0%, and 9972 peptides were identi ed by spectral analysis. 8705 phosphorylated peptides were identi ed. We identi ed a total of 11325 phosphorylation sites on 3448 proteins, of which 10118 sites on 3309 proteins have quantitative information (Table 1, Fig.   2). 3.2 Differential modi cation analysis of OC1 on phosphorylated protein in AD cell model The quanti ed value of the modi ed peptide corresponding to each sample was determined by mass spectrometry quantitative analysis. For each repeated experiment, the ratio of the quantitative values between the two different samples was taken as the comparative group difference expression(Ratio).
Since this project has performed two repeated experiments, for each comparison group,we take the average of the two repeated ratio values as the ratio value of the comparison group, and the coe cient of variation CV of the two repeated Ratio values as the comparison group. When CV-value less than 0.1, the difference expression amount changes by more than 1.2 as a signi cantly up-regulated change threshold,

Sample repeatability test
For biological replicates or technically replicated samples, we examined whether the quantitative results of biological replicates or technical replicates were statistically consistent. Here we use the relative standard deviation(RSD) statistical analysis method to evaluate the quantitative repeatability of the modi cation. As shown in Fig.4, the overall RSD value is small and the quantitative repeatability is good.

Functional enrichment and cluster analysis of differentially modi ed proteins in AD cell model after OC1 administration
Based on the identi cation of all the proteins containing the modi ed site proteins and the screening of the proteins corresponding to the differentially modi ed sites, we performed enrichment analysis and cluster analysis to detect whether the differential modi cations have signi cant enrichment trend in some functional types.

OC1 increases the expression of differentially modi ed proteins associated with calcium channels
In order to detect whether the differential modi cation has a signi cant enrichment trend in certain functional types, we performed GO classi cation, KEGG pathway, and protein domain enrichment analysis on all differentially modi ed proteins identi ed by the screening. For the p-value obtained from the enrichment test,the functional classi cation and pathway of signi cant enrichment of differentially modi ed proteins(P<0.05) were demonstrated by means of a bubble chart. In Fig.5, the vertical axis is the functional classi cation or pathway, and the horizontal axis is the log2 converted value of the ratio of the percentage of the differentially modi ed protein in the functional type compared to the identi ed protein.
It can be seen from the gure that through the three-level enrichment analysis, we have obtained differentially modi ed proteins related to calcium channel activation after OC1 administration (Fig.5) 3.4.2 OC1 increases the expression of differentially modi ed protein p-CaMK (Thr286) related to calcium signaling pathway After enriching the differentially modi ed proteins in different comparison groups, we performed KEGG cluster analysis on them to nd the correlation of the function of the differentially modi ed proteins after OC1 administration. According to the enrichment test P value obtained from the enrichment analysis, the related functions in different groups are brought together to draw a heat map using a hierarchical clustering method. The horizontal direction of the heat map represents different comparison groups, and the vertical direction describes the functions related to differential modi cation and enrichment. Different color blocks indicate the degree of enrichment, red means that the degree of enrichment is strong, and blue means that the degree of enrichment is weak.Compared with the Model(Aβ 1-42 ) group, differentially modi ed proteins were enriched to the Ca 2+ signaling pathway (see Fig. 6A) after OC1 administration. In this pathway we found that the expression of the protein p-CaMK (Thr286) was up-regulated after OC1 administration (see Figure 6B). CaMKII is one of the most abundant kinases in neurons, comprising 1-2% of the total protein concentration. This multifunctional kinase is a major mediator of calcium signaling in neurons, which can further affect the substrate Synapsin1(Syn1) (Ser603). Participate in the remodeling of synapses, mediate the release of transmitters and participate in learning and memory.

CaMKII(Thr286) and Syn1(Ser603)Protein modi cation motif analysis
The protein motif analysis calculates the regularity of the amino acid sequence in the phosphorylation modi cation site by counting the regularity of the amino acid sequence before and after all phosphorylation sites in the sample. This analysis revealed the sequence characteristics of the modi ed sites of the target proteins p-CaMKII(Thr286) and p-Syn1(Ser603), then provide evidence for subsequent discovery of related proteins (Table 3).

GO secondary annotation classi cation
To understand the modi ed proteins identi ed and quanti ed in the data, we have detailed comments on the functions and characteristics of these proteins. Gene Ontology(GO) is a gene theory that expresses various properties of genes and gene products. GO annotations are divided into three broad categories: Biological Process, Cellular Component,and Molecular Function,which explain the biological effects of proteins from different perspectives. We calculated the distribution of the differentially modi ed loci corresponding proteins p-CaMKII(Thr286) and p-Syn1(Ser603) in the GO secondary annotation.As shown in Fig.7, p-CaMKII(Thr286) and p-Syn1(Ser603) are mainly located in the cell junction and synapse (Fig.7) 3.7 Effects of OC1 on Aβ-induced changes in p-CaMK (Thr286), p-Synapsin1(Ser603)/ Synapsin1, Synaptophysin, and Synaptotagmin-1 Our results demonstrate that OC1 protects PC12 cells from Aβ 1-42 -induced cytotoxicity and improves cell viability. Monosialyltetrahexose gangliosides are a class of glycosphingolipids that contain sialic acid widely on the cell membranes of vertebrates and are closely related to the formation, elongation and differentiation of synapses. And choose monosialic acid tetrahexose ganglioside(GM1)(50μmol/L) as the positive drug(Wei Guan et al., 2017;Hu Xufeng et al., 2017). Western blot assay applied to detect the effect of OC1 on p-CaMKII(Thr286) and vesicle-related proteins in AD cell model. The results showed that compared with the control group, the expression of p-CaMK (Thr286) and vesicle proteins p-Synapsin1(Ser603)/Synapsin1, Synaptophysin,and Synaptotagmin-1 in the Aβ1-42 group were signi cantly reduced(P<0.05); on the contrary, the expression of p-Synapsin1(Ser603)/Synapsin1 and Synaptophysin increased by about 20.59% and 31.70%, respectively, in the OC1(2μg/mL) group, compared with the Aβ1-42 group, Moreover, the expressions of p-CaMK (Thr286) and vesicular proteins p-Synapsin1(Ser603)/Synapsin1, Synaptophysin, and Synaptotagmin-1 increased by approximately 30.67%, 48.53%, 139.5%, and 84.48% (P<0.05); Compared with the Aβ 1-42 group, the expression of p-CaMK (Thr286), p-Synapsin1(Ser603)/Synapsin1, Synaptophysin, and Synaptotagmin-1 were increased by approximately 32.00%, 38.24%, 102.3%, and 91.37% (P <0.05), show in Fig. 8.

Discussion
Aβ 1-42 is a metabolite of Amyloid precursor protein(APP). Recent studies have indicated that the nonbrillar soluble oligomeric form of amyloid β protein (sAβ) rather than insoluble amyloid brils or plaques is the cause of the synaptic dysfunction and cognitive defects associated with AD. It has been reported that autophagy plays an important role in the generation and metabolism of Aβ, and thus its malfunction may lead to the progress of AD (Toneff T et al., 2013). Therefore, Aβ-induced apoptosis in PC12 cells was a reliable cellular toxicity model for AD related studies in vitro. In our experiment, the 10µmol/L Aβ 1-42 induced cell model was established, and it was found that pre-treatment with 10μg/mL of OC1 could effectively improve cell viability and reduce the change of cell morphology.
Protein phosphorylation is one of the most important posttranslational modi cations in living cells and is involved in the regulation of a large number of biological processes, such as cell cycle, signal transduction, differentiation, proliferation, metabolism, and autophagy. Whether it is normal physiological processes or cytotoxic protein aggregation in neurodegenerative diseases, it is related to posttranslational modi cation of proteins. Phosphorylation is the most widely studied post-translational modi cation of proteins, besides of phosphorylation of serine, threonine, and tyrosine. Phosphorylation is involved in almost all cellular processes and plays a critical role in the release of neurotransmitters. Advances in proteomic technology with a rapid development of liquid chromatographytandem mass spectrometry (LC/MS-MS) have provided a great opportunity to study phosphorylation events in detail. In this experiment, TMT labeling and phosphorylation modi cation enrichment technology and highresolution liquid chromatography-mass spectrometry combined with quantitative proteomics were used to study the effect of verbascoside on Aβ-induced Rat adrenal medulla pheochromocytoma cells(PC12) cellrelated vesicle protein expression (Zhiqiang W et al., 2018;Erickson B K et al., 2014;Weekes M et al., 2014).
In the present study, OC1 can increase the expression of p-CaMKII(Thr286) protein in AD cell model. AD patients and is primarily due to disrupting synaptic plasticity, Ca 2+ homeostasis and signaling pathways such as Ca 2+ /calmodulin-dependent protein kinase kinase. As an important second messenger, Ca 2+ enrolls in various physiological and biochemical processes in the body. The Ca 2+ /CaM-CaMKII signaling pathway plays an important role in the formation and maintenance of learning and memory in the central nervous system. CaM is an important signaling molecule in the cell.It combines with Ca 2+ to form a Ca 2+ /CaM complex. It activates downstream CaMKII to participate in various physiological effects such as learning and memory (Sun Jingran et al., 2018). CaMKII as a multifunctional serine/threonine protein kinase. The unique properties, keypositions, and complex regulation of CaMKII allow it to participate in important synaptic functions, including the synthesis and release of neurotransmitters. (MIN D Y et al., 2012). Ca 2+ -CaM induces the conformational change of CaMKII, and activated CaMKII can activate the intranuclear transcription mechanism of neurons, thereby regulating the synthesis of substrates downstream and downstream of neurons ( Liyan Z et al., 2018).
Mo04020 Calcium signaling pathway was obtained from KEGG signal pathway enrichment and clustering. Calcium enters cells and activates CaMKII protein. Synaptic vesicles are a very small and highly specialized organelle whose function is known to store and release neurotransmitters. SynapsinI is a nerve terminal-speci c phosphoprotein involved in synaptic transmission, neural development, and neurite outgrowth (Zhu L et al., 2018;Liu Xiao et al., 2017). After being activated by CaMKII, Phosphorylated Synapsin I causes SVs to dissociate from the actin cytoskeleton (Chen X et al., 2018). Synaptophysin is a synaptic vesicle glycoprotein that is widely expressed on presynaptic membranes and strongly associated with synaptic plasticity. Elevated Synaptophysin levels have a positive effect on consolidation of learning and memory after injury. During the release of neurotransmitters, it will move the vesicles to the plasma membrane and promote the formation of SNARE complexes (Südhof,ThomasC,2013). Calcium-binding protein Synaptagmin is a protein located in the synaptic vesicle membrane.After being induced by Ca 2+ , it can be inserted into the presynaptic membrane or act on the fusion pore (Ciani L et al., 2015;Sudhof, T. C., 2015;Jiong Tang et al., 2016), after which, under the combined in uence of the SNARE complex and Ca 2+ , SVs anchor and fuse to the synaptic membrane, enabling the release of neurotransmitters into the synaptic cleft. The experimental Western Blot results showed that OC1 can increase the expression of p-CaMKII(Thr286),p-Synapsin1(Ser603), synaptophysin and synaptotagmin-1 in PC12 cells after Aβ 1-42 intervention. These results indicated that OC1 can affect the release of neurotransmitters by increasing the expression of synaptic vesicle-related proteins.

Conclusion
Through phosphorylation modi cation enrichment technology and quantitative proteomics analysis results, we can conclude safely that verbascoside can increase the expression of CaMKII protein threonine 286 phosphorylation by affecting the amount of calcium ions, which in turn triggers downstream synaptic vesicle correlation. In the future, we will continue to carry out in-vivo experimental veri cation to further improve the research on the role of verbascoside on the nervous system. The datasets generated and analysed during the current study are not publicly available due the research group will continue to conduct in-depth data mining and veri cation, but are available from the corresponding author on reasonable request.

Competing interests
No scramble for interest in this section Funding This study was supported by Inner Mongolia Natural Science Foundation Project (2018MS08026) and State Key Laboratory of Natural Pharmaceutical Active Substances and Functions (GTZK201710). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Authors' contributions GL and HX participated in designing, data collection, analysis, interpretation, and manuscript writing; SW, LZ, CG and YG participated in data analysis, results interpretation; ZD, XD and LF guided the conduct of the experiment and the revision and polishing of the manuscript. All authors have read and approved the nal version of the manuscript.