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Mar. Drugs 2017, 15(1), 20; doi:10.3390/md15010020

Article
Quantitative Proteomic Profiling of Tachyplesin I Targets in U251 Gliomaspheres
Xuan Li , Jianguo Dai , Yongjun Tang, Lulu Li and Gang Jin *
School of Applied Chemistry and Biotechnology, Shenzhen Polytechnic, No. 2190 Liuxian Road, Nanshan District, Shenzhen 518055, Guangdong, China
*
Correspondence:
These authors contributed equally to this work.
Academic Editor: Paul Long
Received: 20 November 2016 / Accepted: 12 January 2017 / Published: 18 January 2017

Abstract

:
Tachyplesin I is a cationic peptide isolated from hemocytes of the horseshoe crab and its anti-tumor activity has been demonstrated in several tumor cells. However, there is limited information providing the global effects and mechanisms of tachyplesin I on glioblastoma multiforme (GBM). Here, by using two complementary proteomic strategies (2D-DIGE and dimethyl isotope labeling-based shotgun proteomics), we explored the effect of tachyplesin I on the proteome of gliomaspheres, a three-dimensional growth model formed by a GBM cell line U251. In total, the expression levels of 192 proteins were found to be significantly altered by tachyplesin I treatment. Gene ontology (GO) analysis revealed that many of them were cytoskeleton proteins and lysosomal acid hydrolases, and the mostly altered biological process was related to cellular metabolism, especially glycolysis. Moreover, we built protein–protein interaction network of these proteins and suggested the important role of DNA topoisomerase 2-alpha (TOP2A) in the signal-transduction cascade of tachyplesin I. In conclusion, we propose that tachyplesin I might down-regulate cathepsins in lysosomes and up-regulate TOP2A to inhibit migration and promote apoptosis in glioma, thus contribute to its anti-tumor function. Our results suggest tachyplesin I is a potential candidate for treatment of glioma.
Keywords:
tachyplesin I; glioblastoma multiforme; cancer stem cell; stable isotope dimethyl labeling; parallel reaction monitoring

1. Introduction

Gliomas, the most common group of primary brain tumors, are subcategorized into astrocytomas, oligodendrogliomas and ependymomas. According to World Health Organization (WHO), glioblastoma multiforme (GBM), the most malignant and lethal form of brain tumor in adults, is a grade IV astrocytoma with very high morbidity and mortality. The disease has a very poor prognosis with short median survival, only about 15 months, despite current multimodal treatment including maximal surgical resection if feasible, followed by a combination of radiotherapy and/or chemotherapy [1]. Therefore, it is imperative to present new and more effective therapeutic interventions to better control GBM.
In fact, the short median survival of GBM is largely ascribed to the inevitable tumor recurrence. Recent research has paid more attention to the existence of glioma stem cells (GSCs), which are a subgroup of tumor cells with properties that resemble those of neural stem cells, and are able to drive tumorigenesis and likely contribute to rapid tumor recurrence [2]. These cells were first described more than ten years ago and have been demonstrated with the capability of multi-lineage differentiation, self-renewal and extensive proliferation [3]. In addition, GSCs can endure and even thrive in stressful tumor conditions, including hypoxia, oxidative stress, inflammation, acidic stress, and low glucose [4]. Moreover, their resistance to conventional therapy and promotion of tumor angiogenesis also influence clinical practice [5,6]. Thus, GSCs provide new insight into the strategy in GBM therapy.
Three-dimensional growth model, a growth sphere formed by cancer stem cells under specific culture conditions in vitro, is a more reasonable model for tumor biology and drug screening in vitro studies [7,8]. Likewise, GSCs also have the characteristic of forming spheres and clinical data show that the rates of existence of gliomaspheres were more prominent in high grade malignant gliomas [9]. Previously, we isolated gliomaspheres from U251 glioma cell lines and tried to apply it for drug screening. We found that there were undifferentiated GSCs and differentiated cancer cells with different differentiation degrees in gliomaspheres, which were similar to the growth state of glioma in vivo [10]. Our previous data showed that gliomaspheres express stem cell biomarkers nestin and CD133, which are certain phenotypes of GSC, and tachyplesin I inhibited the viability and proliferation of gliomaspheres dose dependently, by damaging the plasma membrane and inducing differentiation of GSCs [11]. These findings indicate that tachyplesin I is a potential anti-tumor drug which may be used in GBM therapy.
Tachyplesin I, a cationic peptide with 17 residues (NH2-K-W-C-F-R-V-C-Y-R-G-I-C-Y-I-R-R-C-R-CONH2), was originally isolated from hemocytes of the horseshoe crab (Tachypleus tridentatus) [12]. It has the ability of anti-enzymatic hydrolysis due to two disulfide-stabilized β-hairpins [13]. Several studies have demonstrated that tachyplesin I can inhibit the proliferation and affect the differentiation of tumor cells, such as hepatocarcinoma, gastric adenocarcinoma and leukemia [14,15]. This peptide has also been demonstrated to activate the classic complement pathway to lyse and kill tumor cells and to alter the expression of tumor suppressor genes and oncogenes to induce cell differentiation and reverse the malignant phenotype [16,17]. Most interestingly, the negatively charged components of cancer cells, which are quite different from neutral normal cells, are more vulnerable by the positively charged cationic peptides, including tachyplesin I. The electrostatic attraction between cancer cells and cationic peptides is believed to play a major role in the selective disruption of cancer cell membranes, which avoids traditional mechanism of drug resistance [18].
Although the anti-tumor effect of tachyplesin I has been studied to some extent, the mechanism of anti-tumor activity in GBM is largely unknown. In recent years, proteomics has been shown to be a powerful approach for exploring the molecular mechanisms of anti-tumor drugs. In this study, our primary goal was to identify the changes in protein expression profile of U251 gliomaspheres under the treatment of tachyplesin I, which may help us to better understand the molecular mechanisms underlying potential anti-glioma drugs. Here, both gel-based and shotgun proteomic approaches were performed to gain a higher proteome coverage and better quantification results [19]. Proteomic analysis using two dimension difference gel electrophoresis (2D-DIGE) and stable isotope dimethyl labeling based Liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) revealed that 192 proteins were differentially expressed in U251 gliomaspheres in response to tachyplesin I. Biological involvement of these proteins are further discussed in detail through signaling pathways and protein–protein interaction network analysis. Furthermore, the expression of cathepsins in lysosomes and TOP2A was further validated by Western blot and PRM, due to their important involvement in the anti-tumor activity of tachyplesin I, by inhibiting migration and promote apoptosis of glioma cells, respectively.

2. Results

2.1. Protein Expression Profile of Tachyplesin I Treated U251 Gliomaspheres Using 2D-DIGE Analysis

The 2D-DIGE images, which were scanned at the wavelengths of 488/520, 532/580, and 633/670 nm, visualize the protein expression pattern in the cells (Figure 1A). In the image analysis, 1298 protein spots were detected. Of these, 35 spots with fold change larger than ±1.5 were considered significantly altered in tachyplesin I treated U251 gliomaspheres compared with untreated control (Figure 1B). Among the protein spots that satisfied the statistical criteria, 26 were confidently identified by MALDI-TOF/TOF analysis. Out of 26 identified proteins, 13 were up-regulated while the others were down-regulated in tachyplesin I treated U251 gliomaspheres. Up-regulated proteins were mainly involved in regulation of cell cycle and apoptosis, and cytoskeleton proteins (Table 1). Conversely, down-regulated proteins were involved in glycolysis, response to stimulus and calcium or ion binding (Table 1). Several proteins (Vimentin, Phosphate carrier protein, mitochondrial and Guanine nucleotide-binding protein G(q) subunit alpha) were identified more than once in different location of 2D-DIGE gel, suggesting diverse protein isoforms, such as the occurrence of post-translational modification. Representative images of one up-regulated protein endothelin-converting enzyme 1 (ECE1) and one down-regulated protein alpha-enolase (ENO1) in different dose groups are shown in Figure 1C. Western blot assay was performed to confirm the results obtained from 2D-DIGE experiment and the results were consistent (Figure 1C).

2.2. Relative Quantification Using Dimethyl Labeling Based LC-MS/MS Analysis

Peptide samples from the control, and 10 μg/mL and 40 μg/mL tachyplesin I-treated U251 gliomaspheres were labeled with dimethyl stable isotope tags. To obtain reliable quantification results, we conducted one forward and one reverse dimethyl labeling experiments. A total of 74,240 peptides from 4891 proteins were identified in the forward-labeling samples and 73,892 peptides from 4854 proteins in the reverse-labeling samples (Supplementary Materials Tables S1–S4). In both forward and reverse labeling experiment, the labeled peptides account for more than 99.8% of total identified peptides, indicating a good labeling efficiency. A total of 5737 proteins were reliably quantified in both the forward and reverse labeling experiments, of which 4008 proteins were overlapped (Figure 2B). The protein ratios of L/H and M/H in the forward labeling experiment and protein ratios of M/L and H/L in the reverse labeling experiment indicate the relative abundance of proteins in 10 μg/mL and 40 μg/mL tachyplesin I-treated groups compared to the control. The log2 transformed protein ratios between two different experimental groups all form a symmetric distribution curve with the peak around zero (the original ratio = 1) (Figure 2A), and proteins that were increased or decreased in the forward-labeling experiment were also increased or decreased in the reverse-labeling experiment (Figure 2C), suggesting that there was no bias in the labeling and LC-MS experiments. Only those proteins with fold changes >2 and quantified in both forward and reverse labeling experiments were reported as differentially expressed proteins. Among 4088 proteins, the expression levels of 166 were significantly altered by tachyplesin I treatment. Among them, 55 were up-regulated (Table 2) while 111 proteins were down-regulated (Table 2). Figure 2D shows representative mass spectrometric results for the identification and quantification of the peptide DPDAQPGGELMLGGTDSK from cathepsin D, which clearly reveals the down-regulation of this protein in both sets of experiments.

2.3. Cellular Functions of Differentially Expressed Proteins and Associated Pathways

Systematic gene ontology (GO) analysis of 192 differentially expressed proteins identified from both 2D-DIGE and dimethyl labeling proteomic approaches was performed using PANTHER and DAVID tools. Molecular function analysis revealed that the majority of the differentially expressed proteins demonstrated catalytic (42.93%), binding (26.18%) and structural molecule activities (10.99%) (Figure 3A). The biological processes altered by tachyplesin I treatment were most involved in metabolic processes (30.13%), cellular processes (19.88%), developmental processes (8.43%), localization (8.43%) and biological regulation (8.13%) (Figure 3B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including lysosome pathway (15 proteins), glycosaminoglycan degradation pathway (6 proteins), antigen processing and presentation pathway (8 proteins), DNA replication pathway (5 proteins), type I diabetes mellitus pathway (4 proteins) and glycolysis/gluconeogenesis pathway (4 proteins) are the top pathways altered in response to tachyplesin I treatment (Table 3).

2.4. Tachyplesin I Influences Metabolic Process and Alters the Expressions of Cytoskeleton Proteins

In our study, altered proteins involved in metabolic process occupied major share. Of which, glycolytic/gluconeogenesis enzymes including alpha-enolase (ENO1), gamma-enolase (ENO2), triosephosphate isomerase (TPI1) and phosphoglycerate kinase 1 (PGK1) were found to be down-regulated in response to tachyplesin I treatment. In addition, tachyplesin I treatment on U251 gliomaspheres changed the expression of cytoskeleton proteins. Eighteen out of 192 altered proteins induced by tachyplesin I were classified into cytoskeleton protein class in PANTHER classification system. Several cytoskeleton proteins such as spectrin beta chain, non-erythrocytic 2 (SPTBN2), keratin, type II cytoskeletal 1 (KRT1), keratin, type II cytoskeletal 2 epidermal (KRT2), keratin, type I cytoskeletal 9 (KRT9), keratin, type I cytoskeletal 10 (KRT10), vimentin (VIM), ezrin (EZR), interferon-induced GTP-binding protein Mx1 (MX1), interferon-induced GTP-binding protein Mx2 (MX2), cysteine and glycine-rich protein 1 (CSRP1), elongation factor 1-gamma (EEF1G) and tubulin beta-2B chain (TUBB2B) were down-regulated (Table 2) while neurofilament light polypeptide (NEFL), nestin (NES), kinesin-like protein KIF11 (KIF11), tropomyosin alpha-4 chain (TPM4), dystonin (DST) and LIM and cysteine-rich domains protein 1 (LMCD1) were observed with up-regulation (Table 2). To some extent, all these downstream effects of tachyplesin I contribute to its anti-tumor activity.

2.5. Tachyplesin I Reduces Expressions of Several Lysosomal Acid Hydrolases

As shown in Figure 4A, consistent with the results of proteomic analysis, protein level of lysosomal protective protein (CTSA) was verified to be down-regulated by tachyplesin I using Western blot. Further, other family members of cathepsins, including cathepsin B (CTSB) and cathepsin D (CTSD), as well as cathepsin A (CTSA) were analyzed by PRM mass spectrometry with three technical replicates. For each protein, two unique peptides were selected and monitored for quantification. The skyline software was used to extract the peak areas (area under the curve, AUC) of six to seven strongest transition ions for each peptide (Supplementary Materials Table S5). The normalized sum AUC of all the transitions for each peptide are showed in Figure 4B, which demonstrates that two unique peptides derived from the same protein have a consistent trend, and variations among different technical replicates are small. The results of PRM analysis showed that tachyplesin I down-regulated the levels of CTSA, CTSB and CTSD, which are consistent with dimethyl labeling results.

2.6. Protein-Protein Interaction Network of Differentially Expressed Proteins

Protein–protein interaction (PPI) network was established based on the total 192 differentially expressed proteins related to tachyplesin I treatment, including 26 proteins found in 2D-DIGE analysis and 166 proteins found in dimethyl labeling-based LC-MS analysis. Among them, 180 proteins could connect into a network through direct interaction or an intermediate partner at the PPI level (Figure 5A). Interestingly, DNA topoisomerase 2-alpha (TOP2A) seemed to be the crucial protein in the effects of tachyplesin I as it has the most numerous connections and forms the most complex link with other proteins in the signal network (Figure 5B).

2.7. Confirmation of the Involvement of TOP2A in the Effects of Tachyplesin I and Correlation with Clinical Prognosis in TCGA Database

Western blot result (Figure 5C) showed that the expression level of TOP2A was dose-dependent increased after treatment of tachyplesin I, which was consistent with the result of dimethyl labeling based LC-MS/MS analysis (Table 2). At the same time, the expression level of TOP2A was also checked by PRM analysis. As shown in Figure 5D, after tachyplesin I treatment, the expression level of TOP2A was up-regulated, which verified the data obtained from dimethyl labeling based quantification. Then, we used cBioPortal tool to analyze the relationship between the mRNA transcript level of TOP2A and clinical prognosis of GBM patients based on TCGA database to examine the effects of tachyplesin I by targeting on TOP2A. As shown in Figure 5E, patients with alterations in TOP2A at mRNA transcript level have a better prognosis compared with those without alterations in TOP2A. The analysis showed a significantly better overall and disease-free survival of patients with over-expression of TOP2A.

3. Discussion

More and more studies have shown that certain cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells [20]. Tachyplesin I, which is isolated from hemocytes of the horseshoe crab, has been identified as a member of AMPs and exhibits cytotoxic activity against cancer cells. However, it is uncertain why only some types of AMPs get kill cancer cells, while others not. Besides, whether the molecular mechanisms underlying the antitumor and antimicrobial activities are the same or not remains unclear. Through this study we aim to identify the protein targets of tachyplesin I in U251 gliomaspheres by carrying out a large-scale proteome analysis, which can help us to better understand the molecular mechanisms underlying AMPs as potential anti-glioma drugs.
In this study, gel-based 2D-DIGE and stable isotope dimethyl labeling based LC-MS/MS analysis were combined to reveal the alteration in proteome of U251 gliomaspheres treated with tachyplesin I. A total of 192 differentially expressed proteins were identified, most of which are involved in the cellular process of metabolism, especially glycolysis process, and many proteins are localized as cytoskeleton proteins and lysosomal acid hydrolases. Especially, the expression level of some proteins of interest was validated by PRM, a high-resolution method first published in 2012 and had several potential advantages over traditional approach [21]. For example, PRM spectra are highly specific as a result of all the product ions of a peptide are recorded to confirm peptide identity, while traditional MRM analysis can only monitor one transition of a precursor peptide at a time. Moreover, high-resolution of the orbitrap mass analyzer can separate co-eluted background ions, thus increasing selectivity [22].
One of the hallmarks of tumor cells is the preference of glycolysis over oxidative phosphorylation as the main source of energy. Although glycolysis yield less ATP compared to oxidative phosphorylation with the same amount of beginning materials, tumor cells overcome this disadvantage by increasing the up-take of glucose, thus facilitates a higher rate of glycolysis [23]. Studies have showed that glycolysis plays a role in the invasion activity of glioma cells and is becoming a potential drug target [24]. In this study, glycolytic/gluconeogenesis enzymes including alpha-enolase (ENO1), gamma-enolase (ENO2), triosephosphate isomerase (TPI1) and phosphoglycerate kinase 1 (PGK1) were down-regulated in response to tachyplesin I treatment, indicating that tachyplesin I may disrupt the normal energy metabolism process in gliomaspheres through reduced glycolysis, thus contributing to its anti-tumor effect.
Uncontrolled and invasive proliferation is one feature of grade IV glioma, and in order to block and restrain mitotic division, cytoskeleton has been a time-honored target in cancer chemotherapy [25]. In this study, tachyplesin I treatment on U251 gliomaspheres altered the expression of 18 cytoskeleton proteins as classified by PANTHER classification system. Among them, vimentin and ezrin, which are known to be involved in the regulation of metastasis, were down-regulated under the treatment of tachyplesin I, suggesting that cytoskeleton are influenced by tachyplesin I, thus contributes to its anti-tumor activity.
Out of 192 altered proteins, 15 are lysosomal acid hydrolases, including proteases, glycosidases, sulfatases, lipases and so on. In addition, DAVID pathway classification system revealed lysosome as the most significantly altered pathway. More and more experimental evidences suggest that tumor invasion and metastasis are associated with alterations in lysosomes and increased expression of the lysosomal proteases termed cathepsins [26]. In this study, cathepsins consist of cathepsin A, B and D were down-regulated in response to tachyplesin I treatment. Cathepsin A, also called lysosomal protective protein, is a serine carboxypeptidase implicated in autophagy. It induces tumor cell dissemination and a significant increase in cathepsin A activity in lysates of metastatic lesions of malignant tumor was observed compared to primary focus lysates [27]. Cathepsin B is a lysosomal cysteine protease of the papain family of enzymes that function as an endopeptidase and an exopeptidase [28]. Cathepsin D, an aspartic protease resides in membrane of lysosomes, is involved in autophagy and apoptosis pathways [29]. Interestingly, it has been shown that cathepsin B and D play an important role in human glioma progression and invasion [30]. The expression and enzyme activity of cathepsin B and D gradually increased in high-grade glioblastoma. Inhibition of cathepsin B or D activity attenuates extracellular matrix degradation thus reduces migration of glioma cells [31]. Our data showed that the levels of these cathepsins were significantly decreased in tachyplesin I-treated gliomaspheres compared with untreated cells. All those evidences indicate the potential of tachyplesin I as a therapeutic agent for glioma by targeting the lysosomal activity.
In further PPI analysis of differentially expressed proteins, DNA topoisomerase 2-alpha (TOP2A) was shown to be the possible critical target protein of tachyplesin I. TOP2A is a nuclear enzyme for regulation of DNA topology and replication. TOP2A was discovered to be the target of many anti-tumor drugs which had already been widely used in clinic. Previous reports have shown that DNA damage and fragmentation induced by covalent binding of TOP2A to DNA, and forced expression of TOP2A in cells triggered the apoptotic cell death [32,33]. In addition, the TOP2A level has a close relationship with the activity of these anti-tumor drugs and a high level of TOP2A is the foundation of drug susceptibility. Meanwhile, decreased level, altered phosphorylation or mutation of TOP2A could induce the loss of anti-tumor drug target and develop multiple drug resistance (MDR), which has been confirmed in atypical MDR studies with many cell lines [34,35]. Our results showed that there was an increased TOP2A level in U251 gliomaspheres treated with tachyplesin I and it suggest the possible synergistic effect with TOP2A-targeting drugs, combination of which may be more effective on targeted goals and improve chemotherapy effect.

4. Material and Methods

4.1. Cell Culture and Treatment with Tachyplesin I

U251 human glioma cells were obtained from the Chinese Academy of Sciences Cell Band (Shanghai, China) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The U251 cells in the logarithmic growth phase were thoroughly dissociated to prepare single-cell suspensions. Cell suspensions were washed twice in PBS and resuspended in Neurobasal-A medium with 1× B27 plus 50 ng/mL basic fibroblast growth factor (bFGF) and 50 ng/mL epidermal growth factor (EGF). After 7 days culture, clones of different morphological types were collected. The obtained cells which exhibited certain glioma stem cell phenotypes [11] were cultured as gliomaspheres and passaged every 7 days, based on sphere size.
Tachyplesin I was synthesized by Hanyu Bioengineering Company (Shenzhen, China) with a purity of >95%. Concentrations of tachyplesin I for cell exposure were determined by cell viability assay as described previously [11]. The second generation gliomaspheres were treated with 0, 10, 40 and 80 μg/mL of tachyplesin I for 24 h, and then cells were centrifuged and collected.

4.2. CyDye Minimal Labeling of Protein Samples and 2D-DIGE Electrophoresis

Proteins were extracted from gliomaspheres treated with 0, 10, 40 and 80 μg/mL of tachyplesin I using the lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS and 30 mM Tris-HCl. The concentrations of proteins were determined with the 2-D Quant kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions. Then an equal amount (25 μg) of each protein sample was minimally labeled with Cy3 or Cy5 fluorescent dyes (GE Healthcare) according to the manufacturer’s recommended protocols and the internal standard, resulting from pooling equal aliquots of all experimental samples, was labeled with Cy2.
Six differently pooled samples (Table 4), which comprised equal amounts of Cy3- and Cy5-labeled protein samples and Cy2-labeled internal standard, were then separated by first dimension of isoelectric focusing using 24 cm IPG strips (pH 3–11, nonlinear gradient, GE Healthcare), followed by second dimension separation into 12.5% SDS-PAGE gels. Gels were then scanned with different channels for Cy2-, Cy3-, and Cy5-labeled proteins, using a Typhoon Trio Variable Mode Imager (GE Healthcare). The resulting 18 maps were imported into DeCyder 2D v6.5 (GE Healthcare) for statistical analysis. Each gel was separately processed by the Differential In-gel Analysis (DIA) module for spot detection, background subtraction and in-gel normalization before processed by the Biological Variation Analysis (BVA) module for spot matching and intercomparison across the six gels. Student’s t-test was used to analyze the significance of protein spots between two groups, and one way ANOVA was subsequently used to assess the biological significance among all the experimental groups. Statistically significant spots (p < 0.05) with an average ratio ≥1.5 or ≤−1.5 were chosen for protein identification.

4.3. In-Gel Digestion and Protein Identification by MALDI-TOF/TOF

For identification of spots of interest, a gel was prepared by separating 1 mg of unlabeled proteins pooled from all the samples. The gel was stained by Coomassie Brilliant Blue G-250 and destained by water to reveal the protein spots. After matching to the analytical DIGE gel, each spot of interest was manually excised from the gel and put into a 1.5 mL tube, followed by thorough decoloration with 50% acetonitrile in 25 mM ammonium bicarbonate and dehydration in 100% acetonitrile. Then each gel piece was digested overnight at 37 °C by trypsin in 25 mM ammonium bicarbonate buffer. Peptides were extracted from each gel piece, desalted, and identified by an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) according to previously described [20].

4.4. Dimethyl Labeling of Protein Samples

Cells were lysed with a lysis buffer containing 4% SDS, 100 mM Tris, pH 8.0 and 1× protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MS, USA). One milligram protein from each sample was reduced with 5 mM dithiothreitol, alkylated with 15 mM iodoacetamide, and precipitated by methanol and chloroform [36]. The resulting pellets were resuspended in lysis buffer containing 8 M urea, 0.1 M Tris-HCl, pH 8.5 and the concentration of urea was diluted to below 2 M before overnight digestion with trypsin (Promega, Madison, WI, USA).
Dimethyl labeling was performed on-column according to Nature Protocols by Boersema P.J. et al. [37] with minor modifications. Briefly, acidified peptide samples were loaded into SepPak columns (Waters, Milford, MA, USA) after the columns were activated by methanol, 80% acetonitrile in 0.1% trifluoroacetic acid (TFA), and conditioned by 0.1% TFA. After desalting, the samples were labeled separately by passing the columns with CH2O and NaBH3CN (light), CD2O and NaBH3CN (medium) and CD2O and NaBD3CN (heavy) (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at room temperature. Labeling scheme was shown in Table 5. Then the differentially labeled samples were eluted from the columns, mixed and dried by Speedvac (Labconco, Kansas, MO, USA).

4.5. High pH Fractionation of Peptides and LC-MS/MS Analysis by Obitrap

The dimethyl-labeled sample was resuspended in 1% formic acid (FA), loaded into SepPak column, and fractionated into five fractions by eluting the peptides with 3%, 6%, 9%, 15% and 80% (vol/vol) acetonitrile in 5 mM ammonium formate (pH 10.0), sequentially. After lyophilization in Speedvac, samples were resuspended in 0.1% FA and analyzed by a Q-Exactive orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1000 (Thermo Fisher Scientific) ultrahigh pressure liquid chromatography (UHPLC). The LC separation system consisted of a trap column (100 μm i.d. × 4 cm) and an analytical column (75 μm i.d. × 20 cm) both packed with 3 μm/120 Å C18 resins (Dr. Maisch HPLC GmbH, Ammerbuch, Germany). The eluting buffers were 0.1% FA in H2O (buffer A) and 0.1% FA in 99.9% ACN (buffer B). The peptides were first loaded onto the trap column and then separated by the analytical column with 50 min gradient from 7% to 22% buffer B followed by 10 min gradient from 22% to 35% buffer B at a flow rate of 300 nL/min. MS data was acquired in data dependent acquisition (DDA) mode. Survey full scan MS spectra (m/z 350–1550) were acquired in the Orbitrap with resolution of 70,000, target automatic gain control (AGC) value of 3 × 106, and maximum injection time of 100 ms. Dynamic exclusion for scanned presursors was employed for 60 s. After each MS scan, the 10 most intense precursor ions (z ≥ 2) were sequentially isolated and fragmented by higher-energy collisional dissociation (HCD) using normalized energy 27% with an AGC target of 1 × 105 and a maxima injection time of 50 ms at 17,500 resolution.
Raw data were searched through UniProt Homo sapiens protein database containing 70,076 sequence entries via Sequest HT algorithm with the following parameters: two missed cleavage sites by trypsin, 10 ppm mass tolerance for precursors, 0.02 Da mass tolerance for fragments, and carbamidomethylation (+57.021 Da) of cysteineas static modifications. Moreover, the following dynamic modifications were also set: oxidation of methionine (+15.995 Da), deamidation of asparagine or glutarnine (+0.984 Da), and dimethylation for light-labeled (+28.031 Da) or medium-labeled (+32.056 Da) or heavy-labeled (+36.076 Da) lysine, and N-terminus. All the identified peptides were filtered by FDR <0.01 as reliable identification. Protein Discoverer was used for relative quantification. Differentially expressed proteins were considered for ratios ≤0.5 (down-regulated) and ≥2 (up-regulated).

4.6. Bioinformatic Analysis

The function reports of the candidate proteins whose expression was altered in U251 gliomaspheres due to the effect of tachyplesin I treatment were obtained from the UniProt database (http://www.uniprot.org/) and the protein list of UniProt IDs was input into the PANTHER classification system (http://pantherdb.org/) for GO analysis according to their molecular functions and biological processes. The relevant signaling pathways highly associated with the effect of tachyplesin I treatment on U251 gliomaspheres were identified using DAVID analysis (https://david.ncifcrf.gov/). The protein–protein interaction network of all the differentially expressed proteins was established using String (http://string-db.org/), and then the data was exported as .net file and imported into pajek software for degree based partition of the proteins in the network. The correlation of the possible key proteins involved in the effects of tachyplesin I in our proteomic analysis with its mRNA transcript level and clinical prognosis in GBM patients based on per TCGA data was analyzed by cBioPortal tools (http://www.cbioportal.org/) [38].

4.7. Parallel Reaction Monitoring (PRM) Mass Spectrometry

We applied PRM to validate the major protein changes observed in the dimethyl labeling analyses. Proteins were extracted from another batch of differently treated U251 gliomaspheres (biological replicate) and digested to peptides. These unlabeled peptides were fractionated and identified as described above with only difference in database searching (no dimethylation as dynamic modifications). For PRM analysis, 2 μg of non-fractionated peptides from each group were separated using the same LC system. Linear gradient ranging from 4% to 35% buffer B over 60 min was used. For each target protein, two unique precursor peptide ions were monitored in the inclusion list. The settings for MS full scan were the same as in the DDA mode with only different in m/z scan range (300–900). The following MS/MS PRM scan parameters were set: orbitrap resolution of 35,000, AGC target value of 5 × 105, auto maximum IT, isolation window of 2 m/z, HCD collision energy of 27, and starting mass of m/z 110. The PRM raw files were analysed using Skyline [39] to extract the peak areas of six to seven most intense transitions for each peptide. Then the data was imported to GraphPad for statistical analysis. Differences between two groups were analyzed by the Student’s t-test and statistical significance was considered when p < 0.05.

4.8. Western Blot Assay

Total proteins were extracted from different groups of U251 gliomaspheres with the same treatment as described in the DIGE analysis, and protein concentrations were quantified by BCA kit. Western blot procedures were carried out as we previously described [40], with minor modifications. Namely, after boiling for 5 min with loading buffer, the same amount of proteins from each groups were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with mouse monoclonal anti-ECE-1 antibody (sc-376017, Santa Cruz, CA, USA), mouse monoclonal anti-alpha-enolase (sc-101513, Santa Cruz, CA, USA), mouse monoclonal anti-cathepsin A (sc-73766, Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (sc-32233, Santa Cruz, CA, USA) at 1:500 dilution. The immunoblots were developed by incubation with goat anti-mouse IgG-HRP (sc-2005, Santa Cruz, CA, USA) as the secondary antibody followed by ECL detection (GE Healthcare).

5. Conclusions

In our study, we combined a gel-based 2D-DIGE approach and a dimethyl labeling LC-MS-based shotgun proteomic strategy to identify the proteome expression alterations in U251 gliomaspheres treated with different doses of tachyplesin I. Our results demonstrate complementary advantages of these two techniques. We show that tachyplesin I alters the cellular metabolism, especially glycolysis process and changes the expression of several cytoskeleton proteins and lysosomal acid hydrolases. Moreover, the important role of DNA topoisomerase 2-alpha (TOP2A) in the signal cascades of tachyplesin I was suggested. Further, parallel reaction monitoring (PRM) mass spectrometry confirmed that the major protein of lysosomal acid hydrolases including cathepsin A, cathepsin B and cathepsin D were down-regulated and the possible target-related protein TOP2A was up-regulated by tachyplesin I treatment. In conclusion, we propose that tachyplesin I may down-regulate cathepsins in lysome and up-regulate TOP2A to inhibit migration and promote apoptosis in glioma, thus contributing to its anti-tumor activity. Further work including functional analyses is needed to elucidate the mode of action of tachyplesin I in tumor cells. As far as we know, there is no previous report that reveals the effect of tachyplesin I on proteome of gliomaspheres and our findings imply that tachyplesin I could serve as a promising candidate in the combined therapy against glioma.

Supplementary Materials

The following are available online at www.mdpi.com/1660-3397/15/1/20/s1, Table S1: Total identified peptides information in the forward dimethyl labeling experiment, Table S2: Total identified peptides information in the reverse dimethyl labeling experiment, Table S3: Total identified proteins in the forward dimethyl labeling experiment, Table S4: Total identified proteins in the reverse dimethyl labeling experiment, Table S5: Transitions obtained in Parallel Reaction Monitoring (PRM).

Acknowledgments

This work was supported by the Shenzhen Science and Technology Development Fund Project (No. JCYJ20130331151022276 and GGJS20130331152344401), GDUHTP (2011 and 2013), GDPRSFS (2012), the Project of Guangdong Science and Technology Plan (No. 2014A020217021) and the National Natural Science Foundation of China (No. 31272474).

Author Contributions

Gang Jin conceived and designed the experiments; Xuan Li performed the experiments and wrote the paper; Jianguo Dai contributed reagents/materials/analysis tools and revised the manuscript; Yongjun Tang analyzed the data; and Lulu Li cultured the cells.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Two dimension difference gel electrophoresis (2D-DIGE) analysis of U251 gliomaspheres after treated with tachyplesin I. (A) Representative scanned 2D-DIGE images of Cy2, Cy3, and Cy5, and their overlay derived from a single gel; (B) Representative 2D-DIGE protein profiles with the protein spots marked as differentially regulated in U251 gliomaspheres treated with tachyplesin I. Information about the proteins corresponding to the spot numbers is listed in Table 1; (C) The expression levels of endothelin-converting enzyme 1 (ECE1) and alpha-enolase (ENO1) in U251 gliomaspheres treated by 0, 10, 40 and 80 μg/mL of tachyplesin I for 24 h are visualized by protein abundance maps (first panel), 2-DE images (second panel), three-dimensional spot images (third panel) and validated by Western blot (bottom panel). GAPDH was used as a loading control.
Figure 1. Two dimension difference gel electrophoresis (2D-DIGE) analysis of U251 gliomaspheres after treated with tachyplesin I. (A) Representative scanned 2D-DIGE images of Cy2, Cy3, and Cy5, and their overlay derived from a single gel; (B) Representative 2D-DIGE protein profiles with the protein spots marked as differentially regulated in U251 gliomaspheres treated with tachyplesin I. Information about the proteins corresponding to the spot numbers is listed in Table 1; (C) The expression levels of endothelin-converting enzyme 1 (ECE1) and alpha-enolase (ENO1) in U251 gliomaspheres treated by 0, 10, 40 and 80 μg/mL of tachyplesin I for 24 h are visualized by protein abundance maps (first panel), 2-DE images (second panel), three-dimensional spot images (third panel) and validated by Western blot (bottom panel). GAPDH was used as a loading control.
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Figure 2. Dimethyl labeling based Liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis of U251 gliomaspheres after treated with tachyplesin I. (A) Distribution of quantified protein log2 ratios; (B) A Venn diagram shows the number of proteins identified in either forward or reverse labeling experiment, as well as the overlap between them; (C) A scatter plot showing the forward (y-axis) and reverse (x-axis) dimethyl labeling log2 ratios for the 4008 proteins that were identified and quantified in both experiment, the left panel corresponds to 10 μg/mL group versus control, the right panel corresponds to 40 μg/mL group versus control. The values for each protein are shown as a blue diamond; (D) Representative mass spectrometric image revealing the tachyplesin I-induced down regulation of cathepsin D. Shown are the MS for the peptide DPDAQPGGELMLGGTDSK of cathepsin D from the forward (left panel) and reverse (right panel) dimethyl labeling samples.
Figure 2. Dimethyl labeling based Liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis of U251 gliomaspheres after treated with tachyplesin I. (A) Distribution of quantified protein log2 ratios; (B) A Venn diagram shows the number of proteins identified in either forward or reverse labeling experiment, as well as the overlap between them; (C) A scatter plot showing the forward (y-axis) and reverse (x-axis) dimethyl labeling log2 ratios for the 4008 proteins that were identified and quantified in both experiment, the left panel corresponds to 10 μg/mL group versus control, the right panel corresponds to 40 μg/mL group versus control. The values for each protein are shown as a blue diamond; (D) Representative mass spectrometric image revealing the tachyplesin I-induced down regulation of cathepsin D. Shown are the MS for the peptide DPDAQPGGELMLGGTDSK of cathepsin D from the forward (left panel) and reverse (right panel) dimethyl labeling samples.
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Figure 3. Gene ontology analysis of 192 differentially expressed proteins. The significant (p ≤ 0.001) molecular functions (A) and biological processes (B) are presented in the pie chart.
Figure 3. Gene ontology analysis of 192 differentially expressed proteins. The significant (p ≤ 0.001) molecular functions (A) and biological processes (B) are presented in the pie chart.
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Figure 4. Tachyplesin I reduces expressions of several lysosomal acid hydrolases in U251 gliomaspheres. (A) Expression of cathepsin A (CTSA) was validated by Western blot. GAPDH was used as a loading control; (B) Expressions of CTSA, cathepsin B (CTSB) and cathepsin D (CTSD) were validated by PRM mass spectrometry. The quantification for two peptides per protein in different dose groups is presented.
Figure 4. Tachyplesin I reduces expressions of several lysosomal acid hydrolases in U251 gliomaspheres. (A) Expression of cathepsin A (CTSA) was validated by Western blot. GAPDH was used as a loading control; (B) Expressions of CTSA, cathepsin B (CTSB) and cathepsin D (CTSD) were validated by PRM mass spectrometry. The quantification for two peptides per protein in different dose groups is presented.
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Figure 5. Role of DNA topoisomerase 2-alpha (TOP2A) in protein–protein interaction (PPI) map of tachyplesin I. (A) The constructed minimum PPI network of tachyplesin I containing 192 differentially expressed proteins found in 2D-DIGE and dimethyl labeling-based LC-MS analysis; (B) Degree distribution map of the proteins in PPI network of tachyplesin I. The proteins are shown as round dots and different colors were only related to degree in the network. TOP2A exhibited to have the biggest degree among all differentially expressed proteins; (C) Expression of TOP2A was validated by Western blot. GAPDH was used as a loading control; (D) Expression of TOP2A was validated by PRM mass spectrometry. The quantification for two peptides per protein in different dose groups is presented; (E) The overall survival (left panel) and the disease-free survival (right panel) of glioma cases with or without alterations in TOP2A. The red curves in the Kaplan–Meier plots includes cases with alterations in TOP2A, the blue curves includes cases without alterations in TOP2A.
Figure 5. Role of DNA topoisomerase 2-alpha (TOP2A) in protein–protein interaction (PPI) map of tachyplesin I. (A) The constructed minimum PPI network of tachyplesin I containing 192 differentially expressed proteins found in 2D-DIGE and dimethyl labeling-based LC-MS analysis; (B) Degree distribution map of the proteins in PPI network of tachyplesin I. The proteins are shown as round dots and different colors were only related to degree in the network. TOP2A exhibited to have the biggest degree among all differentially expressed proteins; (C) Expression of TOP2A was validated by Western blot. GAPDH was used as a loading control; (D) Expression of TOP2A was validated by PRM mass spectrometry. The quantification for two peptides per protein in different dose groups is presented; (E) The overall survival (left panel) and the disease-free survival (right panel) of glioma cases with or without alterations in TOP2A. The red curves in the Kaplan–Meier plots includes cases with alterations in TOP2A, the blue curves includes cases without alterations in TOP2A.
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Table 1. Regulated proteins of tachyplesin I treated U251 gliomaspheres in the 2D-DIGE study.
Table 1. Regulated proteins of tachyplesin I treated U251 gliomaspheres in the 2D-DIGE study.
Up-Regulated Proteins of Tachyplesin I Treated U251 Gliomaspheres in the 2D-DIGE Study
No. aGene NameUniprot IDProtein NameMascot ScorePeptidesProtein MWpI ValueRatio/p Value bRatio/p Value b
10 vs. 0 c40 vs. 0 c
Regulation of cell cycle or apoptosis d
2PHGDHO43175d-3-phosphoglycerate dehydrogenase104357,3566.31.54/0.0031.69/0.017
5MSH2P43246DNA mismatch repair protein Msh2932104,7435.81.21/0.0091.77/0.035
19SESN3P58005Sestrin-3201457,2916.31.52/0.0362.01/0.027
31CKAP2Q8WWK9Cytoskeleton-associated protein 276776,9879.4ND e1.52/0.039
35ECE1P42892Endothelin-converting enzyme 146187,1645.91.58/0.0383.31/0.026
Cytoskeletal protein d
3VIMP08670Vimentin4071253,6764.9ND1.64/0.022
4EEF1GP26641Elongation factor 1-gamma 47250,4296.3ND1.51/0.005
9EZRP15311Ezrin168369,4845.91.03/0.0241.63/0.046
10VIMP08670Vimentin5241553,6764.9ND1.58/0.041
Protein biosynthesis d
6EEF2P13639Elongation factor 2 60196,2466.4ND1.55/0.044
21PPIAP62937Peptidyl-prolyl cis-trans isomerase A 1951618,22991.26/0.0171.52/0.028
Transport d
23SLC25A3F8VVM2Phosphate carrier protein, mitochondrial90536,1619.3ND1.72/0.034
25SLC25A3F8VVM2Phosphate carrier protein, mitochondrial183936,1619.3ND1.63/0.016
Down-Regulated Proteins of Tachyplesin I Treated U251 Gliomaspheres in the 2D-DIGE Study
Calcium or iron ion binding protein d
7EPS15P42566Epidermal growth factor receptor substrate 15109598,6565.1ND e−1.88/0.004
13P4HA1P13674Prolyl 4-hydroxylase subunit alpha-1 86561,2965.6−1.51/0.007−2.37/0.045
Regulation of cell apoptosis or proliferation d
12ANXA5P08758Annexin A5 2731335,9714.8ND−1.74/0.033
20GSTP1P09211Glutathione S-transferase P 3392123,5695.3ND−1.66/0.001
33COL4A3BPQ9Y5P4Collagen type IV alpha-3-binding protein250770,8355.5ND−1.64/0.033
34ARHGDIAP52565Rho GDP-dissociation inhibitor 1 2361623,2504.9−1.23/0.037−2.64/0.047
Response to stimulus d
14GNAQP50148Guanine nucleotide-binding protein G(q) subunit alpha193942,1425.7ND−1.61/0.037
16GNAQP50148Guanine nucleotide-binding protein G(q) subunit alpha 2941242,1425.7ND−1.59/0.047
28GNAQP50148Guanine nucleotide-binding protein G(q) subunit alpha 182742,1425.7−1.33/0.028−1.53/0.036
Glycolysis/Gluconeogenesis d
15ENO1P06733Alpha-enolase40347,4817.7−1.04/0.005−1.92/0.054
17PGK1P00558Phosphoglycerate kinase 1209744,9859.2−1.68/0.025−2.89/0.051
30TPI1P60174Triosephosphate isomerase3751531,0575.6−1.17/0.048−1.88/0.028
Ribosomal protein d
18RPSAP0886540S ribosomal protein SA171932,9474.6−1.49/0.036−1.89/0.027
a No.—The numbers correspond to the spot numbers indicated in Figure 1B; b Average ratios of spot abundance of tachyplesin I-treated samples relative to the control, represent data from three separate experiments and student’s t test p values are given as a measure of confidence for the ratio of each spot measured; c 0: control group; 10: 10 μg/mL dose group; 40: 40 μg/mL dose group; d Functional categories according to Gene ontology and panther biological process annotations; e ND, not detected or p value > 0.5.
Table 2. List of proteins with altered expression in U251 gliomaspheres after treatment of tachyplesin I using dimethyl labeling quantitative proteomic analysis.
Table 2. List of proteins with altered expression in U251 gliomaspheres after treatment of tachyplesin I using dimethyl labeling quantitative proteomic analysis.
The 55 Up-Regulated Proteins Expressed More Than 2 Folds (<1% FDR)
Gene NameUniprot IDProtein NameCoverage (%) aUnique Peptides a10 vs. 0 Ratio b40 vs. 0 Ratio bProtein Class c
ForwardReverseForwardReverse
SPP1P10451Osteopontin33.1254.9612.94312.8769.484cytokine
ITGB3P05106Integrin beta-35.2034.7545.95313.27928.714receptor, extracellular matrix glycoprotein
EPS8Q12929Epidermal growth factor receptor kinase substrate 84.0134.4032.5492.2202.305transmembrane receptor regulatory/adaptor protein
MCM5B1AHB1DNA helicase5.5033.3042.0223.8012.194DNA helicase
DKK1O94907Dickkopf-related protein 111.2843.2672.2783.8852.732developmental protein, growth factor activity
MCM4P33991DNA replication licensing factor MCM47.7643.2482.1195.1893.723DNA binding protein
NUSAP1Q9BXS6Nucleolar and spindle-associated protein 118.1452.6612.2933.1213.405microtubule-associated protein
DHFRP00374Dihydrofolate reductase24.0642.6162.1682.3982.472reductase
TOP2AP11388DNA topoisomerase 2-alpha12.93122.4902.9163.2592.582DNA topoisomerase, enzyme modulator
MKI67A0A087WV66Antigen KI-6712.66242.3961.8702.6882.047regulation of cell proliferation
TFRCP02786Transferrin receptor protein 135.79232.3232.3982.9062.879receptor
AIM1Q9Y4K1Absent in melanoma 1 protein21.53252.2982.3294.0365.403carbohydrate binding protein
ECE1P42892Endothelin-converting enzyme 115.1982.2602.6713.5574.070metalloprotease
SYNJ2O15056Synaptojanin-28.76112.2392.5015.3494.740phosphatase
KIF11P52732Kinesin-like protein KIF112.3722.1992.2742.4692.182microtubule binding motor protein
DSTQ03001Dystonin23.49212.1131.5962.6142.051non-motor actin binding protein
UPP1Q16831Uridine phosphorylase 146.45112.0782.7122.2093.577phosphorylase
IGFBP5P24593Insulin-like growth factor-binding protein 520.5952.0702.0604.8164.967cell communication
RRM2P31350Ribonucleoside-diphosphate reductase subunit M234.45112.0301.9812.2862.423reductase
CD70P32970CD70 antigen35.2361.9952.1673.7253.808cell communication
MDKE9PPJ5Midkine (Fragment)27.4821.9352.5383.5694.336cytokine
HMGCS1Q01581Hydroxymethylglutaryl-CoA synthase, cytoplasmic42.88171.9291.9393.3224.339transferase, lyase
DCLK1Q5VZY9Serine/threonine-protein kinase DCLK110.7431.8993.2224.7218.819non-receptor serine/threonine protein kinase
MCM7P33993DNA replication licensing factor MCM713.2171.8791.9632.4512.470DNA helicase
PODXLO00592Podocalyxin2.3311.8702.2342.4742.856regulation of adhesion and cell morphology
MCM2H0Y8E6DNA replication licensing factor MCM2 (Fragment)8.2561.8651.5132.1672.664DNA helicase
LPLP06858Lipoprotein lipase34.11121.8482.0784.1364.351storage protein
VSNL1P62760Visinin-like protein 124.0841.8052.2603.2312.926cell communication
MCM6Q14566DNA replication licensing factor MCM69.0141.7652.2082.6063.031DNA helicase
GPC1P35052Glypican-133.69141.7451.6652.8613.347cell division and growth regulation
TACC3Q9Y6A5Transforming acidic coiled-coil-containing protein 33.2221.7352.0682.5632.322cytoskeleton
TNCP24821Tenascin40.1651.7301.8032.3272.112signaling molecule
PLATP00750Tissue-type plasminogen activator24.73121.7293.4758.26713.172receptor, calmodulin
GATMP50440Glycine amidinotransferase, mitochondrial28.6191.7041.7932.5912.740catalyze creatine biosynthesis
SERPINE1P05121Plasminogen activator inhibitor 121.1471.6951.4484.5393.853serine protease inhibitor
LMCD1Q9NZU5LIM and cysteine-rich domains protein 138.63101.6811.6862.3882.243structural protein
TYMSP04818Thymidylate synthase19.1741.6752.7522.1513.228methyltransferase
ITGA3P26006Integrin alpha-321.41191.6711.7292.8472.944receptor, integrin
ANLNQ9NQW6Actin-binding protein anillin3.9131.6662.2352.3083.143actin binding protein
ANXA2P07355Annexin A281.42341.6171.6102.0232.100fatty acid metabolic process
MACF1H3BPE1Microtubule-actin cross-linking factor 1, isoforms 1/2/3/529.641531.6101.6012.0182.000non-motor actin binding protein
TPM4P67936Tropomyosin alpha-4 chain48.7981.5342.1262.8782.893actin binding motor protein
ACTN4K7EJH8Alpha-actinin-4 (Fragment)68.6811.5262.4112.2513.153non-motor actin binding protein
TRIM9Q9C026E3 ubiquitin-protein ligase TRIM94.2331.5081.1432.0772.109ubiquitin-protein ligase
LDLRP01130Low-density lipoprotein receptor6.6351.4981.2022.2662.096receptor, extracellular matrix glycoprotein
SDCBPO00560Syntenin-128.5241.4951.4902.5813.361membrane trafficking regulatory protein
TFP02787Serotransferrin45.13271.4221.5262.2242.216transfer/carrier protein
TENM2H7BYZ1Teneurin-213.86241.4221.7352.0462.501receptor, membrane-bound signaling molecule
NESP48681Nestin58.61781.3961.3632.1402.167structural protein
THY1E9PIM6Thy-1 membrane glycoprotein (Fragment)25.6631.3601.4682.4322.131membrane glycoprotein
NEFLP07196Neurofilament light polypeptide47.88281.2131.1822.1212.010structural protein
CLSTN1Q5SR54Calsyntenin-1 (Fragment)4.3531.1811.2012.3382.552cell adhesion molecule, calcium-binding protein
ECI2A0A0C4DGA2Enoyl-CoA delta isomerase 2, mitochondrial40.38111.1481.2622.2392.894transfer/carrier protein, enzyme modulator
PTPREP23469Receptor-type tyrosine-protein phosphatase epsilon11.2950.9392.0693.2823.385receptor, protein phosphatase
LRRC16AQ5VZK9Leucine-rich repeat-containing protein 16A1.902ND1.4082.2813.321transcription cofactor
The 111 Down-Regulated Proteins Expressed Less Than 0.5 Folds (<1% FDR)
OASLQ156462′-5′-oligoadenylate synthase-like protein12.2640.1270.3930.215NDnucleotidyltransferase, defense/immunity protein
OAS2P297282′-5′-oligoadenylate synthase 29.7490.1510.1580.1150.068nucleotidyltransferase, defense/immunity protein
MX1P20591Interferon-induced GTP-binding protein Mx156.50280.1640.1770.1500.130microtubule family cytoskeletal protein
IFI44LQ53G44Interferon-induced protein 44-like39.60130.1790.2060.1890.191immune response
IFI44Q8TCB0Interferon-induced protein 4433.56130.1930.2310.1580.181immune response
CASP1G3V169Caspase19.3540.2090.3250.2350.180regulation of apoptotic process
BTN3A2E9PRR1Butyrophilin subfamily 3 member A2 (Fragment)27.5520.2200.5550.2610.360ubiquitin-protein ligase
INSC9JNR5Insulin (Fragment)7.6110.2280.2310.6340.769growth factor
MX2P20592Interferon-induced GTP-binding protein Mx216.0550.2350.1400.1290.215microtubule family cytoskeletal protein
PARP10E9PPE7Poly [ADP-ribose] polymerase 104.7120.2600.2800.2010.508nucleic acid binding
ISG15A0A096LNZ9Ubiquitin-like protein ISG15 (Fragment)50.3560.2730.2930.2530.264ribosomal protein
TAP1Q03518Antigen peptide transporter 129.08150.2870.3850.2880.288ATP-binding cassette (ABC) transporter
IFIT3O14879Interferon-induced protein with tetratricopeptide repeats 348.57180.2880.3010.2610.258RNA binding
IFIT2P09913Interferon-induced protein with tetratricopeptide repeats 230.08110.2940.2910.2120.250RNA binding
IFIT1P09914Interferon-induced protein with tetratricopeptide repeats 145.82160.3000.3210.2960.301RNA binding
KRT10P13645Keratin, type I cytoskeletal 1030.14130.3010.3140.4830.541structural protein
DDX58O95786Probable ATP-dependent RNA helicase DDX5841.73370.3070.3070.2780.265helicase, hydrolase
BLOC1S1G8JLQ3Biogenesis of lysosome-related organelles complex 1 subunit 150.6730.3080.4610.2960.417transcription factor
TRIM21P19474E3 ubiquitin-protein ligase TRIM217.7930.3100.3820.2590.340ubiquitin-protein ligase
OAS3Q9Y6K52′-5′-oligoadenylate synthase 330.08290.3160.3060.2460.237nucleotidyltransferase, defense/immunity protein
SLC4A4Q9Y6R1Electrogenic sodium bicarbonate cotransporter 19.6480.3230.2900.1640.214transporter
TAPBPO15533Tapasin25.0070.3240.3970.3180.333immunoglobulin receptor superfamily
KRT1P04264Keratin, type II cytoskeletal 136.49180.3250.2730.5500.447structural protein
DTX3LQ8TDB6E3 ubiquitin-protein ligase DTX3L25.81120.3260.4260.3750.377ubiquitin-protein ligase
TAP2Q03519Antigen peptide transporter 222.16100.3500.3500.2800.270ATP-binding cassette (ABC) transporter
GBP1P32455Interferon-induced guanylate-binding protein 128.38130.3620.3460.2960.202heterotrimeric G-protein
KRT9P35527Keratin, type I cytoskeletal 935.47150.3630.4090.6320.759structural protein
AGTRAPQ6RW13Type-1 angiotensin II receptor-associated protein13.8410.3830.4790.5570.604response to hypoxia
PARP9Q8IXQ6Poly [ADP-ribose] polymerase 916.28120.3870.3920.3950.359nucleic acid binding
HLA-BP30466HLA class I histocompatibility antigen, B-18 alpha chain57.7310.3960.4140.3070.344immunoglobulin receptor superfamily
IRF9Q00978Interferon regulatory factor 97.3830.4050.5880.3180.377immune response
C19orf66Q9NUL5UPF0515 protein C19orf6616.1530.4050.4800.2140.395no function identified yet
NT5EP215895′-nucleotidase48.08250.4080.4280.3530.361nucleotide phosphatase
STAT1P42224Signal transducer and activator of transcription 1-alpha/beta53.33360.4080.4220.3690.381transcription factor, nucleic acid binding
KRT2P35908Keratin, type II cytoskeletal 2 epidermal7.8230.4110.3290.4280.471structural protein
SP100P23497Nuclear autoantigen Sp-1009.5660.4120.4380.3520.363HMG box transcription factor, signaling molecule
B2MP61769Beta-2-microglobulin37.8240.4190.4130.3690.332major histocompatibility complex antigen
ALBA0A0C4DGB6Serum albumin16.8990.4270.4550.6680.654transfer/carrier protein
BANF1O75531Barrier-to-autointegration factor34.8320.4290.4670.3080.408DNA binding, DNA integration
IFIT5Q13325Interferon-induced protein with tetratricopeptide repeats 519.7170.4380.4970.4770.435RNA-binding
ERAP2Q6P179Endoplasmic reticulum aminopeptidase 26.2550.4610.5270.3710.457metalloprotease
HLA-AP01892HLA class I histocompatibility antigen, A-2 alpha chain64.38150.4650.5090.4150.447immunoglobulin receptor superfamily
NDRG1Q92597Protein NDRG127.4160.4680.5410.2600.246stress-responsive protein
STAT2P52630Signal transducer and activator of transcription 210.9350.4780.6170.3470.442transcription factor, nucleic acid binding
HLA-EP13747HLA class I histocompatibility antigen, alpha chain E24.0220.4790.3190.4720.433immunoglobulin receptor superfamily
ATP6V0CP27449V-type proton ATPase 16 kDa proteolipid subunit11.6110.4840.3530.9060.805hydrolase, ATP synthase
UCHL3P15374Ubiquitin carboxyl-terminal hydrolase isozyme L315.6520.4920.4590.5580.220cysteine protease
EPN2F6PQP6Epsin-2 (Fragment)19.5670.4960.5760.2620.295endocytosis
DBIP07108Acyl-CoA-binding protein65.5260.5010.5020.2630.148transfer/carrier protein
SP110G5E9C0SP110 nuclear body protein, isoform CRA_b5.4820.5060.4730.4640.439HMG box transcription factor, signaling molecule
TCEAL3Q969E4Transcription elongation factor A protein-like 316.5020.5070.4780.3540.307transcription factor
LGALS3BPQ08380Galectin-3-binding protein39.83190.5070.5330.4270.457receptor, serine protease
UBE2L6O14933Ubiquitin/ISG15-conjugating enzyme E2 L659.4850.5170.4070.3820.317ligase
SMYD2Q9NRG4N-lysine methyltransferase SMYD27.3930.5190.6690.3270.243transcription cofactor
TREX1Q9NSU2Three-prime repair exonuclease 17.8620.5260.4630.4820.389catalytic activityi
AK4P27144Adenylate kinase 4, mitochondrial49.3380.5290.5000.3810.422nucleotide kinase
FAM96BJ3KS95Mitotic spindle-associated MMXD complex subunit MIP18 (Fragment)23.5820.5390.4210.4520.473iron-sulfur cluster assembly
DPP7Q9UHL4Dipeptidyl peptidase 235.37120.5410.5810.3650.431serine protease
PMLP29590Protein PML33.79220.5450.5580.4240.392activator
AGAP20933N(4)-(beta-N-acetylglucosaminyl)-l-asparaginase24.8650.5510.6350.4150.490protease
EPHA2P29317Ephrin type-A receptor 219.67150.5550.5230.3860.395nervous system development
SERPINI1Q99574Neuroserpin8.7830.5640.7570.2570.217serine protease inhibitor
PAPSS2O95340Bifunctional 3′-phosphoadenosine 5′-phosphosulfate synthase 237.30180.5670.5440.3360.276nucleotidyltransferase
IDUAP35475Alpha-l-iduronidase31.85160.5720.6050.4160.479glycosidase
GLAP06280Alpha-galactosidase A25.6480.5740.6250.4640.435glycosidase, hydrolase
SGSHP51688N-sulphoglucosamine sulphohydrolase29.68110.5780.5150.3530.410hydrolase
GAAP10253Lysosomal alpha-glucosidase24.37190.5840.6570.4260.461glucosidase
CHSY3Q70JA7Chondroitin sulfate synthase 36.9260.5870.4830.3610.320glycosyltransferase
ACP5K7EIP0Tartrate-resistant acid phosphatase type 5 (Fragment)36.5410.5870.5440.3130.246glycosylated monomeric metalloprotein enzyme
PSMB8P28062Proteasome subunit beta type-839.1380.5900.5520.4550.493endopeptidase activity
SPTBN2O15020Spectrin beta chain, non-erythrocytic 24.3520.5930.7380.3890.358non-motor actin binding protein
PGM2L1Q6PCE3Glucose 1,6-bisphosphate synthase48.07300.5980.6270.4920.448glycosyltransferase, mutase
SAMD9LQ8IVG5Sterile alpha motif domain-containing protein 9-like4.6760.6110.5470.4890.416regulation of protein catabolic process
CSTBP04080Cystatin-B45.9230.6150.6800.3280.380cysteine protease inhibitor
LGMNQ99538Legumain10.3940.6180.6360.4830.499cysteine protease
CPQQ9Y646Carboxypeptidase Q20.5570.6200.6310.4060.452carboxypeptidase activity
CTSAP10619Lysosomal protective protein18.7590.6260.6670.4180.422serine protease
NAGAP17050Alpha-N-acetylgalactosaminidase11.9230.6260.6480.4800.287deacetylase
ENO2P09104Gamma-enolase60.83110.6310.6760.4360.494lyase
GALNSP34059N-acetylgalactosamine-6-sulfatase8.6250.6330.6870.4360.377hydrolase
KCTD12Q96CX2BTB/POZ domain-containing protein KCTD1233.23110.6340.6240.4460.480enzyme modulator
GOLIM4O00461Golgi integral membrane protein 418.25110.6380.6710.3910.416transport
NMRK1B3KN26Nicotinamide riboside kinase 112.2610.6410.5270.4220.402kinase
RNASET2D6REQ6Ribonuclease T219.2740.6430.5450.3980.400endoribonuclease activity
TUBB2BQ9BVA1Tubulin beta-2B chain74.1610.6430.5450.4240.318tubulin
MTAPQ13126S-methyl-5′-thioadenosine phosphorylase71.38150.6450.6830.4840.492phosphorylase
NAGLUP54802Alpha-N-acetylglucosaminidase26.11130.6460.7060.4660.478glycosidase, hydrolase
TXNIPQ9H3M7Thioredoxin-interacting protein16.1160.6500.4410.4790.351transcription regulation, oxidative stress mediator
BCAR3O75815Breast cancer anti-estrogen resistance protein 37.8840.6520.2850.1620.272guanine-nucleotide releasing factor
GUSBP08236Beta-glucuronidase26.42160.6780.6490.4950.452galactosidase
PGK1P00558Phosphoglycerate kinase 184.41310.6860.6470.4610.437carbohydrate kinase
H6PDO95479GDH/6PGL endoplasmic bifunctional protein35.65230.7080.7290.4770.483dehydrogenase
CSRP1P21291Cysteine and glycine-rich protein 164.2590.7110.6700.4260.427actin family cytoskeletal protein
CPVLQ9H3G5Probable serine carboxypeptidase CPVL21.2290.7110.6400.4800.453serine protease
NNMTP40261Nicotinamide N-methyltransferase56.06100.7130.6670.3350.333methyltransferase
EXTL3O43909Exostosin-like 315.34130.7370.8070.4330.472glycosyltransferase
VLDLRP98155Very low-density lipoprotein receptor8.4860.7380.6990.4600.478receptor, extracellular matrix glycoprotein
MMP14P50281Matrix metalloproteinase-1419.76110.7480.8000.3440.361hydrolase, metalloprotease, protease
OSTF1Q92882Osteoclast-stimulating factor 153.7490.7500.6490.4590.463signal transduction
AKAP2Q9Y2D5A-kinase anchor protein 215.8370.7510.7730.4100.468regulation of cell cycle, apoptosis process
SIAEQ9HAT2Sialate O-acetylesterase12.0550.7690.7250.3460.294esterase
MRC2Q9UBG0C-type mannose receptor 211.36130.7780.8670.3790.454receptor
IDSP22304Iduronate 2-sulfatase23.8290.7840.8120.4250.465hydrolase
CNTNAP1P78357Contactin-associated protein 12.0220.7920.6380.4030.436transporter, membrane-bound signaling molecule, receptor
AKR1C3S4R3Z2Aldo-keto reductase family 1 member C36.6710.8280.6450.3050.337reductase
AMDHD2Q9Y303Putative N-acetylglucosamine-6-phosphate deacetylase8.0720.8400.5480.4650.473deacetylase
MANBAO00462Beta-mannosidase6.6030.9010.7990.3560.482galactosidase
SH3BP5LQ7L8J4SH3 domain-binding protein 5-like5.0920.9120.9310.3540.411protein kinase inhibitor
LRP1Q07954Prolow-density lipoprotein receptor-related protein 10.6231.0680.6170.3810.478receptor, extracellular matrix glycoprotein
ATF7IPF5GYR7Activating transcription factor 7-interacting protein 1 (Fragment)9.381ND0.4470.4410.146transcription regulation
VPS29Q9UBQ0Vacuolar protein sorting-associated protein 2956.041ND0.9220.4730.474vesicle coat protein
a The values of coverage and unique peptides are based on forward labeling result; b Ratios: Spot abundance of tachyplesin I-treated samples relative to the control; 0: control group; 10: 10 μg/mL dose group; 40: 40 μg/mL dose group; forward: forward labeling group; reverse: reverse labeling group; c Functional categories according to Gene ontology and panther biological process annotations.
Table 3. List of altered KEGG pathways with tachyplesin I treatment and their p-values identified by bioinformatic analysis using DAVID (p < 0.1).
Table 3. List of altered KEGG pathways with tachyplesin I treatment and their p-values identified by bioinformatic analysis using DAVID (p < 0.1).
Pathwaysp ValueDifferentially Expressed Proteins Involved in This Pathway
Lysosome1.11 × 10−8SGSH, AGA, NAGLU, GUSB, LGMN, ACP5, CTSA, MANBA, ATP6V0C, GLA, IDS, GALNS, NAGA, GAA, IDUA
Glycosaminoglycan degradation2.53 × 10−5SGSH, NAGLU, IDS, GUSB, GALNS, IDUA
Antigen processing and presentation5.99 × 10−4TAP2, LGMN, TAP1, HLA-A, HLA-B, HLA-E, TAPBP, B2M
DNA replication3.51 × 10−3MCM7, MCM2, MCM4, MCM5, MCM6
Type I diabetes mellitus3.75 × 10−2INS, HLA-A, HLA-B, HLA-E
Glycolysis/Gluconeogenesis8.93 × 10−2TPI1, ENO2, PGK1, ENO1
Table 4. Labeling scheme of DIGE for U251 gliomaspheres protein.
Table 4. Labeling scheme of DIGE for U251 gliomaspheres protein.
Gel No.Cy2Cy3Cy5
Gel 01StandardA1B2
Gel 02StandardB1C3
Gel 03StandardC2D3
Gel 04StandardD2A2
Gel 05StandardA3C1
Gel 06StandardB3D1
A: control group; B: 10 μg/mL dose group; C: 40 μg/mL dose group; D: 80 μg/mL dose group; 1–3: three biological repeats in each group.
Table 5. Dimethyl-labeling scheme for U251 gliomaspheres protein.
Table 5. Dimethyl-labeling scheme for U251 gliomaspheres protein.
SamplesForwardReverse
control groupHeavy (H)Light (L)
10 μg/mL dose groupLight (L)Medium (M)
40 μg/mL dose groupMedium (M)Heavy (H)
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