Literature DB >> 34917708

Proteomic dataset: Profiling of glioma C6 and astrocytes rat cell lines before and after co-cultivation.

Artemiy S Silantyev1, Olga N Bukato2, Ivan O Butenko2, Anastasia A Chernysheva1, Olga V Pobeguts2, Alexander E Nosyrev3, Olga I Gurina4.   

Abstract

Human multiforme glioblastoma is characterized by an unfavorable prognosis, low survival rate and extremely limited possibilities for therapy. Rat C6 glioma is an experimental model for the study of glioblastoma growth and invasion. It has been shown that the growth and development of the tumor is accompanied by changes in the surrounding normotypic tissues [1]. These changes create a favorable environment for the development of the tumor and give it an evolutionary advantage [2]. Description of changes occurring in normotypic cells of the body upon their contact with tumor cells is of great interest. We have grown C6 glioma cells and rat astrocytes, as well as astrocyte cells co-cultured together with C6 glioma. We performed proteome-wide LC-MS analysis of these experimental groups. The data includes LC-MS/MS raw files and exported MaxQuant and ProteinPilot search results with fasta. Dataset published in the PRIDE repository project accession PXD026776.
© 2021 The Authors.

Entities:  

Keywords:  Co-culture; Glioma C6; LC-MS/MS; Proteome

Year:  2021        PMID： 34917708      PMCID： PMC8666335          DOI： 10.1016/j.dib.2021.107658

Source DB:  PubMed          Journal:  Data Brief        ISSN： 2352-3409


Specifications Table

Value of the Data

These data are acquired from in vitro interaction model of rat glioma and astrocytes cells. This model is helpful for valuable for researchers interested in cancer proteomics. Dataset covers eleven biological and five technical replicates in control (rat astrocytes and glioma C-6 without co-cultivating and) and in co-cultivated cells. Dataset show the possible mechanisms illustrating how glioma cells can interact with astrocytes cells.

Data Description

For our co-cultivated in vitro model, we used astrocytes and C6 glioma cells. Astrocytes cell lines isolated from rat brain tissue. We analyzed astrocytes in two conditions: before and after co-cultivation. Proteins were assessed in a label-free bottom-up proteomic experiment using IDA approach (i.e. Information Dependent Acquisition) on AB Sciex TripleTOF 6600 Q-TOF mass-spectrometer coupled with LFQ (label-free quantification) approach by MaxQuant software. Dataset covers 165 samples (11 biological and 5 technical replicates) (Table 1).
Table 1

Sample description.

Sample nameDescriptionBiological replicateTechnical replicate
20190704A0001_IDA_1rat astrocytes11
20190704A0001_IDA_2rat astrocytes12
20190704A0001_IDA_3rat astrocytes13
20190704A0001_IDA_4rat astrocytes14
20190704A0001_IDA_5rat astrocytes15
20190704A0002_IDA_1rat astrocytes21
20190704A0002_IDA_2rat astrocytes22
20190704A0002_IDA_3rat astrocytes23
20190704A0002_IDA_4rat astrocytes24
20190704A0002_IDA_5rat astrocytes25
20190704A0003_IDA_1rat astrocytes31
20190704A0003_IDA_2rat astrocytes32
20190704A0003_IDA_3rat astrocytes33
20190704A0003_IDA_4rat astrocytes34
20190704A0003_IDA_5rat astrocytes35
20190704A0004_IDA_1rat astrocytes41
20190704A0004_IDA_2rat astrocytes42
20190704A0004_IDA_3rat astrocytes43
20190704A0004_IDA_4rat astrocytes44
20190704A0004_IDA_5rat astrocytes45
20190704A0005_IDA_1rat astrocytes51
20190704A0005_IDA_2rat astrocytes52
20190704A0005_IDA_3rat astrocytes53
20190704A0005_IDA_4rat astrocytes54
20190704A0005_IDA_5rat astrocytes55
20190704A0006_IDA_1rat astrocytes61
20190704A0006_IDA_2rat astrocytes62
20190704A0006_IDA_3rat astrocytes63
20190704A0006_IDA_4rat astrocytes64
20190704A0006_IDA_5rat astrocytes65
20190704A0007_IDA_1rat astrocytes71
20190704A0007_IDA_2rat astrocytes72
20190704A0007_IDA_3rat astrocytes73
20190704A0007_IDA_4rat astrocytes74
20190704A0007_IDA_5rat astrocytes75
20190704A0008_IDA_1rat astrocytes81
20190704A0008_IDA_2rat astrocytes82
20190704A0008_IDA_3rat astrocytes83
20190704A0008_IDA_4rat astrocytes84
20190704A0008_IDA_5rat astrocytes85
20190704A0009_IDA_1rat astrocytes91
20190704A0009_IDA_2rat astrocytes92
20190704A0009_IDA_3rat astrocytes93
20190704A0009_IDA_4rat astrocytes94
20190704A0009_IDA_5rat astrocytes95
20190704A0010_IDA_1rat astrocytes101
20190704A0010_IDA_2rat astrocytes102
20190704A0010_IDA_3rat astrocytes103
20190704A0010_IDA_4rat astrocytes104
20190704A0010_IDA_5rat astrocytes105
20190704A0011_IDA_1rat astrocytes111
20190704A0011_IDA_2rat astrocytes112
20190704A0011_IDA_3rat astrocytes113
20190704A0011_IDA_4rat astrocytes114
20190704A0011_IDA_5rat astrocytes115
20190627G0001_IDA_1rat glioma C611
20190627G0001_IDA_2rat glioma C612
20190627G0001_IDA_3rat glioma C613
20190627G0001_IDA_4rat glioma C614
20190627G0001_IDA_5rat glioma C615
20190627G0002_IDA_1rat glioma C621
20190627G0002_IDA_2rat glioma C622
20190627G0002_IDA_3rat glioma C623
20190627G0002_IDA_4rat glioma C624
20190627G0002_IDA_5rat glioma C625
20190627G0003_IDA_1rat glioma C631
20190627G0003_IDA_2rat glioma C632
20190627G0003_IDA_3rat glioma C633
20190627G0003_IDA_4rat glioma C634
20190627G0003_IDA_5rat glioma C635
20190627G0004_IDA_1rat glioma C641
20190627G0004_IDA_2rat glioma C642
20190627G0004_IDA_3rat glioma C643
20190627G0004_IDA_4rat glioma C644
20190627G0004_IDA_5rat glioma C645
20190628G0005_IDA_1rat glioma C651
20190628G0005_IDA_2rat glioma C652
20190628G0005_IDA_3rat glioma C653
20190628G0005_IDA_4rat glioma C654
20190628G0005_IDA_5rat glioma C655
20190628G0006_IDA_1rat glioma C661
20190628G0006_IDA_2rat glioma C662
20190628G0006_IDA_3rat glioma C663
20190628G0006_IDA_4rat glioma C664
20190628G0006_IDA_5rat glioma C665
20190628G0007_IDA_1rat glioma C671
20190628G0007_IDA_2rat glioma C672
20190628G0007_IDA_3rat glioma C673
20190628G0007_IDA_4rat glioma C674
20190628G0007_IDA_5rat glioma C675
20190628G0008_IDA_1rat glioma C681
20190628G0008_IDA_2rat glioma C682
20190628G0008_IDA_3rat glioma C683
20190628G0008_IDA_4rat glioma C684
20190628G0008_IDA_5rat glioma C685
20190628G0009_IDA_1rat glioma C691
20190628G0009_IDA_2rat glioma C692
20190628G0009_IDA_3rat glioma C693
20190628G0009_IDA_4rat glioma C694
20190628G0009_IDA_5rat glioma C695
20190628G0010_IDA_1rat glioma C6101
20190628G0010_IDA_2rat glioma C6102
20190628G0010_IDA_3rat glioma C6103
20190628G0010_IDA_4rat glioma C6104
20190628G0010_IDA_5rat glioma C6105
20190628G0011_IDA_1rat glioma C6111
20190628G0011_IDA_2rat glioma C6112
20190628G0011_IDA_3rat glioma C6113
20190628G0011_IDA_4rat glioma C6114
20190628G0011_IDA_5rat glioma C6115
20190708RA0001_IDA_1astrocytes co-cultivation with glioma C611
20190708RA0001_IDA_2astrocytes co-cultivation with glioma C612
20190708RA0001_IDA_3astrocytes co-cultivation with glioma C613
20190708RA0001_IDA_4astrocytes co-cultivation with glioma C614
20190708RA0001_IDA_5astrocytes co-cultivation with glioma C615
20190708RA0002_IDA_1astrocytes co-cultivation with glioma C621
20190708RA0002_IDA_2astrocytes co-cultivation with glioma C622
20190708RA0002_IDA_3astrocytes co-cultivation with glioma C623
20190708RA0002_IDA_4astrocytes co-cultivation with glioma C624
20190708RA0002_IDA_5astrocytes co-cultivation with glioma C625
20190708RA0003_IDA_1astrocytes co-cultivation with glioma C631
20190708RA0003_IDA_2astrocytes co-cultivation with glioma C632
20190708RA0003_IDA_3astrocytes co-cultivation with glioma C633
20190708RA0003_IDA_4astrocytes co-cultivation with glioma C634
20190708RA0003_IDA_5astrocytes co-cultivation with glioma C635
20190708RA0004_IDA_1astrocytes co-cultivation with glioma C641
20190708RA0004_IDA_2astrocytes co-cultivation with glioma C642
20190708RA0004_IDA_3astrocytes co-cultivation with glioma C643
20190708RA0004_IDA_4astrocytes co-cultivation with glioma C644
20190708RA0004_IDA_5astrocytes co-cultivation with glioma C645
20190715RA0005_IDA_1astrocytes co-cultivation with glioma C651
20190715RA0005_IDA_2astrocytes co-cultivation with glioma C652
20190715RA0005_IDA_3astrocytes co-cultivation with glioma C653
20190715RA0005_IDA_4astrocytes co-cultivation with glioma C654
20190715RA0005_IDA_5astrocytes co-cultivation with glioma C655
20190715RA0006_IDA_1astrocytes co-cultivation with glioma C661
20190715RA0006_IDA_2astrocytes co-cultivation with glioma C662
20190715RA0006_IDA_3astrocytes co-cultivation with glioma C663
20190715RA0006_IDA_4astrocytes co-cultivation with glioma C664
20190715RA0006_IDA_5astrocytes co-cultivation with glioma C665
20190715RA0007_IDA_1astrocytes co-cultivation with glioma C671
20190715RA0007_IDA_2astrocytes co-cultivation with glioma C672
20190715RA0007_IDA_3astrocytes co-cultivation with glioma C673
20190715RA0007_IDA_4astrocytes co-cultivation with glioma C674
20190715RA0007_IDA_5astrocytes co-cultivation with glioma C675
20190715RA0008_IDA_1astrocytes co-cultivation with glioma C681
20190715RA0008_IDA_2astrocytes co-cultivation with glioma C682
20190715RA0008_IDA_3astrocytes co-cultivation with glioma C683
20190715RA0008_IDA_4astrocytes co-cultivation with glioma C684
20190715RA0008_IDA_5astrocytes co-cultivation with glioma C685
20190716RA0009_IDA_1astrocytes co-cultivation with glioma C691
20190716RA0009_IDA_2astrocytes co-cultivation with glioma C692
20190716RA0009_IDA_3astrocytes co-cultivation with glioma C693
20190716RA0009_IDA_4astrocytes co-cultivation with glioma C694
20190716RA0009_IDA_5astrocytes co-cultivation with glioma C695
20190716RA0010_IDA_1astrocytes co-cultivation with glioma C6101
20190716RA0010_IDA_2astrocytes co-cultivation with glioma C6102
20190716RA0010_IDA_3astrocytes co-cultivation with glioma C6103
20190716RA0010_IDA_4astrocytes co-cultivation with glioma C6104
20190716RA0010_IDA_5astrocytes co-cultivation with glioma C6105
20190716RA0011_IDA_1astrocytes co-cultivation with glioma C6111
20190716RA0011_IDA_2astrocytes co-cultivation with glioma C6112
20190716RA0011_IDA_3astrocytes co-cultivation with glioma C6113
20190716RA0011_IDA_4astrocytes co-cultivation with glioma C6114
20190716RA0011_IDA_5astrocytes co-cultivation with glioma C6115
Sample description.

Experimental Design, Materials and Methods

Cultivation

C6 glioma and astrocytes cell lines isolated from rat brain tissue were grown in 25 cm2 Corning culture flasks. As the growth medium, we used RPMI-1640 (Thermo Fisher Scientific) with the addition of fetal bovine serum (10% of the total volume of the medium), sodium pyruvate (ml/100 ml of medium), L-glutamine (ml/100 ml of medium) and a solution of an antibiotic (ml/100 ml of medium) containing 10,000 IU/ml of penicillin, 10,000 µg/ml of streptomycin and 25 µg/ml of amphotericin B. The growth medium was changed every 2,3 days. Upon reaching the monolayer, cells were dissociated from the culture vials using a 0.25% trypsin solution followed by 3-fold washing with Dubelco's phosphate-saline buffer. Cell counting was performed in a Goryaev chamber and on a TC20TM Automated Cell Counter (BIO-RAD) automatic cell counter. To obtain samples of co-cultured astrocytes, joint cultivation of astrocytes and C6 glioma cells was performed. For this, astrocytes were seeded in 6-well Corning Costar plates in the amount of 300 thousand cells per well. C6 glioma cells were sown in special inserts at a concentration of 50 thousand cells per well. Co-cultivation was carried out for 10 days. The growth medium was updated every 2,3 days. On the 10th day, the inserts were removed, astrocytes were dissociated from the culture plates, and samples were prepared as described above.

Sample preparation

All reagents used to perform sample preparation were manufactured by Sigma-Aldrich and have a grade of purity of OCP, unless otherwise specified. 15 µl of 10% sodium deoxycholate was added to the resulting cell pellet, after which the pellet was placed on a vortex. Then, 1 µl of nuclease mix (GE Healthcare) was added to the pellet, after which the solution was placed on a vortex and intubated for 1 hour at 4°C. Next, a solution containing 100 µl of 100 mM Tris-HCl, 0.05% sodium deoxycholate, 2.5 mM EDTA and 8 M urea (pH 8) was added to the sample. The solution was incubated for 30 minutes at room temperature (RT). After incubation, the sample was centrifuged at 14,000 rpm for 10 min at 20°C (Eppendorf, Centrifuge 5810). After all, the supernatant was selected. The actual abundance of proteins was measured with the Bradford (ThermoFisher, USA) method for protein quantitation. Next, tris (2-carboxyethyl) phosphine (TCEP) was added to the supernatant to a final concentration of 5 mM. The resulting solution was incubated for 1 h at 37°C. Then, iodoacetamide (IAA) was added to the resulting solution to a final concentration30 mM. The resulting solution was incubated for 30 min in the dark at RT. Finally, TCEP was added to the sample to a final concentration 2.5 mM and the sample was incubated for 20 min at RT. The resulting sample solution was diluted with 6 × volume to a final urea concentration 1.5 M and containing 50 mM Tris-HCl and 0.05% DCNa (pH 8). Trypsin (Promega, MS grade) (1 µg / µl) in a ratio of 1:50 (trypsin: protein) was added to the resulting sample. The resulting solution was incubated for 16 h at 37°C. After tryptic digestion (Promega, USA), a solution of 50% trifluoroacetic acid (Sigma, USA) was added to the sample to pH 2.0. After that samples were centrifuged, and the supernatant was taken. The supernatant was purified by solid phase extraction using Discovery DSC-18 Tube (Supelco) columns according to the manufacturer's protocol. Peptides were eluted from a 1 ml column of buffer containing 50% acetonitrile and 0.1% TFA. The resulting peptide extract was dried on a vacuum concentrator (Labconco) almost to dryness and resuspended in a buffer containing 3% acetonitrile and 0.1% TFA to a final concentration of native protein of 5 µg/ml.

LC-MS/MS analysis

The LC-MS/MS analysis of tryptic peptides was performed using a microflow HPLC system Eksigent Ekspert (Sciex, USA) with autosampler Eksigent Ekspert 400 (Sciex, USA) coupled to AB Sciex Triple TOF 6600 with microflow AB Sciex Duo Spray ion Source (Sciex, USA). The chromatographic separation of the peptides was performed with Eksigent column, 2.7 um, HALO Fused-Core C18, 50 × 0.5 mm 805-10100. Chromatographic separation was performed with the parameters: (parameters). The ion source settings were as follow: the flow rate of nebulizer gas - 10 L/min; the voltage applied to the capillary 4500 V. The mod of obtaining MS spectra is as follows: accumulation time 50 ms, mass/charge range from 100 to 2500 m/z, criteria of preferred selection of ions for isolation and fragmentation - charge 2–5, number parent ions from one MS spectrum - 25, the minimum intensity is 5000, resolution 30,000. After the first analysis, the ions were excluded from the candidate for fragmentation 5 s. The fragmentation parameters were as follows: the accumulation time of the spectra fragmentation - 20 ms, charge/mass ratio from 100 to 2500 m/z, full cycle time 600 ms, resolution 25,000.

Protein identification and quantification analysis

Identification and label-free quantification analysis were performed with MaxQuant software with default settings against a database of all proteins of Rattus norvegicus (Uniprot) of 2020. All proteins in database are reviewed by Swiss-Prot. Human keratins were added to all databases to avoid misinterpretation of contaminating proteins.

Analysis and data processing

The data was processed using the software SCIEX Analyst, SCIEX PeakView, SCIEX ProteinPilot, MaxQuant, RStudio, OmicsBox. All identifications are performed with follow parameters: protein and peptide FDR – 1%, fixed modification – Carbamidomethyl (C), variable modification – oxidation (M), Acetyl (Protein N-term), MS and MSMS mass tolerance – 20 ppm, max number of misscleavages site – 2. Other specific parameters of search in mqpar.xml file.

Results

As a result of the analysis, 1567 proteins for astrocytes (A) were quantified; 1667 for glioma (G); 1684 for co-cultured astrocytes (RA). In total, 2708 proteins were quantified for all three biological groups. The result was considered reliable if in a pairwise comparison between the three biological groups, the signal intensity for the protein was more than doubled and the p-value adjusted by Benjamini-Hochberg was less than 0.05. Thus, a change in the level of 162 proteins (G / A) was reliably established between glioma and astrocytes; between co-cultured astrocytes and glioma 141 protein (RA/G); between co-cultured astrocytes and astrocytes, a change of 70 proteins (RA / A) was found.

Ethics Statement

Animals were not used in the experiment. C6 glioma cells and rat astrocytes were purchased from the American Type Culture Collection (ATCC) and were stored and cultured according to the manufacturer's protocols.

CRediT authorship contribution statement

Artemiy S. Silantyev: Writing – original draft, Formal analysis. Olga N. Bukato: Writing – review & editing, Formal analysis. Ivan O. Butenko: Software. Anastasia A. Chernysheva: Methodology. Olga V. Pobeguts: Methodology, Resources. Alexander E. Nosyrev: Conceptualization. Olga I. Gurina: Resources, Funding acquisition, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
SubjectBiology
Specific subject areaProteomics
Type of dataLC-MS/MS data and identification data
How data were acquiredMicroflow HPLC system Eksigent Ekspert (Sciex, USA) with autosampler Eksigent Ekspert 400 (Sciex, USA) coupled to ABSciex 6600 with a microflow AB Sciex Duo Spray Ion Source (Sciex, USA)
Data formatRaw and analyzed data
Parameters for data collectionWe analyzed glioma C6, rat astrocytes co-cultured with glioma C6, rat astrocytes (control).
Description of data collectionDataset covers 165 samples (11 biological replicates and five technical)
Data source locationFederal State Institution V. P. Serbsky Federal Medical Research Center of Psychiatry and Narcology National Scientific Research Center on Addictions of the Ministry of Healthcare of the Russian Federation, 119002 Moscow, Russia
Data accessibilityThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortiumvia the PRIDE partner repository with the dataset identifier PXD026776. Direct link: https://www.ebi.ac.uk/pride/archive/projects/PXD026776RAW data available on FTP server:http://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD026776/
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