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 interest 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. extraction- trypsin digestion- TMT/iTRAQ label- affinity enrichment- HPLC fractionation- mass spectrometry- database search-bioinformatics

immune function enhancement, and anti-aging (Zhang Qing et al.,2018;Yuan Yating et al., 2016;LIN Huimin et al., 2016). According to reports in the literature, Verbascoside can significantly improve the learning and memory dysfunction of subacute aging mice induced by D-galactose, can regulate the activity of Acetylcholine transferase ( ChAT ) and Acetylcholinesterase ( AchE ) in brain tissue, protect neurons in hippocampal CA1 region of brain tissue, improve brain tissue and immune organs index, and improve brain damage in model mice (Gao Li et al., 2014;Yang WN et al., 2014; Gao L et al., 2014;Peng Xiaoming et al., 2015;Xiong, L et al., 2015;Wu Hongyan et al., 2015). Furthermore, verbascoside can significantly improve the survival rate of neurons after Aβ1-42 injury, increase the expression of Synapsin, and inhibit its apoptosis (Hu Hang et al., 2016;Shiao YJ et al., 2017;Bai, P et al., 2013). In this way, it is concluded that Cistanche tubulosa has a certain application value in the pathogenesis of learning and memory disorders such as Alzheimer's disease(AD).
Neurodegenerative diseases are a group of chronic progressive neurological diseases based on primary neuronal degeneration. Among them, AD has a wide incidence and is one of the more common chronic diseases in modern elderly. 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). The main cause of the disease is thought to be due to excessive accumulation of amyloid β-protein (Aβ) in the brain, overexpression of phosphorylated tubulin (tau-protein) and intracellular neuron. The formation of a large number of fiber entanglements (neurofibrillary tangles, NFTs) (Kenney, K et al., 2018).
The deposition of Aβ in the brain can cause degeneration and death of neurons, especially the polymerization of fibrillar form of Aβ is toxic to nerve cells, by injecting Aβ into the brain of animals or in vitro cultured cells. The establishment of AD animal model and cell model by adding Aβ is one of the commonly used tools for studying the pathogenesis and drug treatment of AD (Tian Xinhong et al., 2017).
Pre-in vitro experiments have initially found that verbascoside can antagonize the apoptosis of neuronal cells induced by Aβ during the pathogenesis of AD, and has a significant effect on the release of neurotransmitters during neuronal information transmission ( 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 used to treat neurological diseases 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.

2.2Experimental methods
Through the organic combination of TMT labeling, high performance liquid chromatography grading technology, phosphorylated peptide modification and mass spectrometry-based quantitative proteomics technology, the phosphorylation modification quantitative characterization in the sample is carried out. the study. Its technical route is shown below:Protein extraction-trypsin digestion-TMT/iTRAQ label-affinity enrichment-HPLC fractionation-mass spectrometry-database searchbioinformatics analysis.

2.3Protein extraction
PC12 cells in logarithmic growth phase were inoculated into 6-well plates at 2×10 5 per well, cultured in an incubator for 24 h, and then treated with Aβ1-42 10 μmol L -1 and OC110 μg·mL -1 for 24 h. Gently scrape the cells and store at -80°C for later use.
Samples were removed and 4 volumes of lysis buffer (8 M urea, 1% protease inhibitor, 1% phosphatase inhibitor) were added and sonicated. After centrifugation at 12,000 rpm for 10 min at 4°C, the cell debris was removed, the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

2.4Trypsin digestion and TMT labeling
Dithiothreitol was added to the protein solution to a final concentration of 5 mM and reduced at 56°C for 30 min. Iodoacetamide was then added to a final concentration of 11 mM and incubated for 15 min at room temperature in the dark. Finally, the urea concentration of the sample is diluted to less than 2 M. Trypsin was added at a mass ratio of 1:50 (pancreatin: protein) and digested overnight at 37°C. Trypsin was added at a mass ratio of 1:100 (pancreatin: protein) and continued to digest for 4 h. The trypsin-digested peptide was desalted with Strata X C18 (Phenomenex) and vacuum-dried. The peptide was solubilized with 0.5 M TEAB and the peptides were labeled according to the TMT kit instructions.

2.5HPLC classification and modification enrichment
The peptides were fractionated using high pH reverse HPLC. Peptide fractionation gradient was 8%-32% acetonitrile, pH 60, 60 min time separation of 60 components, followed by peptides combined into 4 components, the combined components were vacuum freeze-dried. The peptide was then dissolved in an enrichment buffer (50% acetonitrile / 6% trifluoroacetic acid) and the supernatant was transferred to pre-washed IMAC material and placed on a rotary shaker for gentle shaking. After the completion of the incubation, the resin was washed three times with a buffer solution of 50% acetonitrile / 6% trifluoroacetic acid and 30% acetonitrile / 0.1% trifluoroacetic acid. Finally, the modified peptide was eluted with 10% ammonia water, and the eluate was collected and vacuum-dried and drained. After draining, the salt was removed according to the C18 ZipTips instructions, vacuum-dried and drained for liquid-mass spectrometry analysis.

2.6Liquid Chromatography-Mass Spectrometry Analysis
The peptides were dissolved in liquid phase A phase (0.1% (v/v) aqueous formic acid) and separated using an EASY-nLC 1000 ultra high performance liquid system. 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 flow 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 secondary mass spectrometry data was searched using Maxquant (v1.5.2.8).

2.7Mass spectrometry control
As shown in the figure below, 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 identification. 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 identified by mass spectrometry meets quality control requirements.

2.8.1Protein annotation method
Gene Ontology analysis: Gene Ontology analysis, or GO analysis, is a bioinformatics analysis method that can link information between genes and gene products (such as 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 (Kyoto Encyclopedia of Genes and Genomes) 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.

2.8.2Analysis of modified site motifs
Software MoMo, the motif-x algorithm was used to analyze the motif features of the modified sites. Among them, 10 identified phosphorylations from upstream and downstream of each identified modification site were peptide fragments consisting of 6 amino acids from upstream to downstream. The analysis background was 10 upstream and downstream of all potential modification sites in the species ( Phosphorylation is a peptide sequence consisting of 6 amino acids of each of the upstream and downstream. When the number of peptides in a certain characteristic sequence form is greater than 20, and the statistical test P value is less than 0.000001, the characteristic sequence form is considered to be a motif of the modified peptide.

2.8.3Cluster analysis based on protein function enrichment
Cluster analysis based on functional enrichment of differentially modified proteins (or differentially modified proteins with different differential folds) based on different groups was used to study their potential associations and differences in the KEGG pathway. We first collect functional classification information enriched for the protein used and the corresponding enriched P-value values, and then screen out functional classifications that are significantly enriched (P-value < 0.05) in at least one protein group. The P-value data matrix obtained by the screening is first subjected to logarithmic transformation with -log10, and then the transformed data matrix is subjected to Z transformation for each function classification. Finally, the data set obtained after Z transformation is analyzed by hierarchical clustering (European distance, average connected clustering). The clustering relationship is visualized using the heat map drawn by the function heatmap.2 in the R language package gplots.

2.9Western blotting
RIAP buffer was used to lyse PC12 cells, nuclear proteins were extracted, and protein content was quantified by BCA method. Add the sample buffer to the same amount of Horseradish peroxide-labeled goat anti-rabbit polyclonal antibody (1:1000) was incubated for 2 h at room temperature, and exposed to ECL luminescence solution.
After scanning the film, Image J software analyzed the gray scale of the strip and performed semi-quantitative comparison analysis. Using its own gray value correction, the expression level of the protein is expressed by the gray scale ratio of the band gray of the target gene to the β-actin of the housekeeping gene.

Statistics
SPSS22.0 statistical software package was used for statistics, and the results were expressed by s x  . The two groups and multiple groups of samples were compared for univariate analysis of variance. P <0.05 indicates that the difference is statistically significant.

3.11Overview of modification identification
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 identified by spectral analysis. 8705 phosphorylated peptides were identified. We identified a total of 11325 phosphorylation sites on 3448 proteins, of which 10118 sites on 3309 proteins have quantitative information (Table 1,   Fig. 2 Basic statistics of mass spectrometry data results

3.2Protein differential modification analysis
The quantified value of the modified 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 coefficient of variation CV of the two repeated Ratio values as the comparison group. CV value.
When CV-value<0.1, the difference expression amount changes by more than 1.2 as a significantly up-regulated change threshold, and less than 1/1.2 as a significantly down-regulated change threshold.The summary data of all differentially expressed proteins(

3.3Sample 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 modification. Figure 3 is a box plot of the relative standard deviation (RSD) of the quantitative values of the modifications between replicates. The overall RSD value is small and the quantitative repeatability is good (Fig. 4).

3.4Protein modification motif analysis
The protein motif analysis calculates the regularity of the amino acid sequence in the phosphorylation modification 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 modified sites of the target proteins CaMK and Syn1 (Table 3, Fig. 5).  Note: Red indicates that this amino acid is significantly enriched near the modification site, and green indicates that this amino acid is significantly reduced near the modification site.

Protein Notes
In order to thoroughly understand the modified proteins identified and quantified in the data, we have detailed comments on the functions and characteristics of these proteins, from Gene Ontology (GO) and subcellular structure localization.

GO secondary annotation classification
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 modified loci corresponding proteins CaMK and Syn1 in the GO secondary annotation. As shown in Fig. 6, the Model vs control group, oc1 vs con group, oc1 vs mod group, CaMK and Syn1 are mainly located in the cell junction and synapse. (Fig. 6) Fig.6 Statistical distribution map of the corresponding phosphorylated modification sites corresponding to Camk and Syn1 in GO secondary classification

Subcellular structure localization classification
We used professional software to predict and classify the subcellular structures of the differentially modified proteins CaMK and Syn1. As shown in Fig. 7, the Model vs control group, oc1 vs con group, oc1 vs mod group. CaMK and Syn1 are mainly located in cytoplasm, Plasma membrane and nucleus (Fig. 7).

Functional enrichment analysis of protein CaMK corresponding to differential modification sites
Based on the identification of all the proteins containing the modified site proteins and the screening of the proteins corresponding to the differentially modified sites, we performed KEGG pathway enrichment analysis and cluster analysis to detect whether the differential modifications have significant enrichment in some functional types. trend.

KEGG pathway enrichment and cluster analysis
The KEGG pathway obtained by enrichment analysis was screened according to the target protein CaMK (Fig. 8A,8B). After classifying and enriching the differentially modified proteins in different comparison groups, we performed KEGG cluster analysis to obtain the related signal pathway mo04020 Calcium signaling pathway of the differentially modified protein CaMKII in the comparison group. (Fig. 8C)  causing neuronal damage and impaired learning and memory. It is widely used in the study of Aβ damage and toxic effects ( Toneff T et al., 2013). Therefore, we used PC12 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. Synapsin is a major protein on synaptic vesicles, and its ability to regulate neurotransmitter release has been demonstrated in some studies (Zhu L et al., 2018;Liu Xiao et al., 2017). Synapsin l is an important protein that connects vesicles to the cytoskeleton. After being activated by CaMKII, Synapsin l (Ser9) is phosphorylated, causing the vesicles to fall from the cytoskeleton to the plasma membrane to participate in vesicle transport ( Chen X et al., 2018).
Synaptophysin is a synaptic vesicle glycoprotein. 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. It is a positive regulator of neurotransmitter release ( Ciani L et al., 2015;Sudhof, T. C., 2015;Jiong Tang et al., 2016). The experimental Western Blot results showed that OC1 can increase the expression of p-CaMKII (Thr286), p-Synapsin l (Ser9), synaptophysin and synaptotagmin-1 in PC12 cells after Aβ1-42 intervention, which will be used for further prevention and treatment of AD. Provide pharmacological basis.

5.CONCLUSION
Through phosphorylation modification enrichment technology and quantitative proteomics student letter analysis results, 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. The effect of protein expression plays a role in protecting AD cell models and regulating neurotransmitter release.