MS-analysis of SILAC-labeled MYC-driven B lymphoma cells overexpressing miR-17-19b

Micro RNAs (miRNAs) are small non-coding RNAs, which dampen gene expression by repressing translation and/or inducing degradation of target-mRNAs. Although the role of miR-17-19b (a truncated version of miR-17-92 cluster) is well documented in MYC-driven B cell lymphomagenesis, little is known about the function of the cluster in the maintenance of full-blown lymphomas. We employed SILAC-based quantitative proteomics to identify miR-17-19b targets upon a mild overexpression of the cluster in B cell lymphomas, established from λ-MYC transgenic mice. The proteomics data described in detail in this study, whose follow up analysis with MaxQuant algorithm is part of the recent publication (Mihailovich et al., 2015) [1], are deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the accession code PRIDE: PXD002810.


a b s t r a c t
Micro RNAs (miRNAs) are small non-coding RNAs, which dampen gene expression by repressing translation and/or inducing degradation of target-mRNAs. Although the role of miR-17-19b (a truncated version of miR-17-92 cluster) is well documented in MYC-driven B cell lymphomagenesis, little is known about the function of the cluster in the maintenance of full-blown lymphomas. We employed SILAC-based quantitative proteomics to identify miR-17-19b targets upon a mild overexpression of the cluster in B cell lymphomas, established from λ-MYC transgenic mice. The proteomics data described in detail in this study, whose follow up analysis with MaxQuant algorithm is part of the recent publication (Mihailovich et al., 2015) [1]  Possibility of using the dataset for comparative analysis of different murine B cell lymphoma models.

Data
Stable Isotope Labeling by Amino Acid in Cell culture (SILAC [2])-based quantitative proteomics was employed to analyze the impact of the miRNAs on the global protein output. In particular, we analyzed by high-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS) full-blown B lymphoma cells, originated from λ-MYC transgenic mice [3], either with a mild overexpression of miR-17-19b (a truncated version of the miR-17-92 cluster), or -as a control-without overexpression of the cluster. In order to generate control and miRNA-overexpressing cells (miRNA cells), respectively, lymphoma cells were infected with a retroviral vector, empty or containing miR-17-19b (Fig. 1A).
The proteomics data obtained by the MS-analysis of these samples are described in the recent publication [1].

Experimental design, materials and methods
In the "Dir" SILAC experimental setups, miRNA cells were grown in Heavy (H) medium and control cells in Light (L) medium, whereas the SILAC channels were swapped in the Rev experiments. Light and Heavy cells were mixed 1:1, and the extracts were in-solution digested with trypsin. Sample complexity was reduced by peptide fractionation, using isoelectric focusing (IEF). LC-MS/MS was carried out on a LTQ-FT Ultra (Thermo Fisher Scientific). The data described hereby correspond to the SILAC-based quantitative analysis of functional experiments performed with two different clones (clones 1 and 2), and control experiment (wild type vs. wild type). See Table 1 and Fig. 1B.

Murine tumor cell line
Mouse B lymphoma cell line (clone ♯2567) was established through in vitro culture of primary lymphomas isolated from λ-MYC transgenic mice [3]. Tumors were cultured in B cell medium (DMEM Table 1 List and description of all LC-MS/MS data acquired.

Cloning and retroviral gene transduction
The GFP containing expression vector pMIG was used for the generation of the plasmid pMIG-miR-17-19b. Fragment miR-17-19b, a truncated version of miR-17-92, was amplified by PCR from mouse genomic DNA (FW_BamHI_miR-17: 5'-CGG GAT CCG TCA GAA TAA TGT CAA AGT GCT-3'; RV_XhoI_-miR-19b: 5'-CCG CTC GAG CAC TAC CAC AGT CAG TTT TGC AT-3'), and inserted into expression vector pMIG by BamHI-XhoI digestion. A spin infection of B lymphoma cells was performed with 2 Â 10 5 cells per well. Retroviruses were produced by Phoenix cells, which were previously transfected with pMIG and pMIG-miR-17-19b plasmids. The efficiency of transduction was evaluated by flow cytometric analysis for GFP positive cells 48 h post-infection, and subsequently GFP-sorted. We performed two independent rounds of sorting for control and miRNA cells, obtaining two different cell populations, which we refer to as clones 1 and 2. The experiments have been performed within the first ten cell passages post-infection.

SILAC labeling
Control and miRNA cells were grown in SILAC media (lysine-and arginine-free DMEM/Ham's F12 (1:1), 10% dialyzed fetal bovine serum (FBS, Invitrogen)), supplemented with 1 mM non-essential amino acids (Gibco), 100 U/ml of penicillin and streptomycin (Lonza), 1 mM Na-pyruvate (Gibco), 2 mM Glutamine (Lonza) and 50 mM β-mercaptoethanol (Gibco)). "Heavy" and "Light" media were obtained by adding 0.146 g/L 13 C 6 , 15 N 2 L-Lysine and 0.84 g/L 13 C 6 15 N 4 L-Arginine (Sigma) or the corresponding non-labeled amino acids, respectively to the SILAC media. Growth in SILAC media was carried out for eight duplications, to ensure complete protein labeling. For the Dir experiments miRNA cells were labeled with heavy medium, and control cells with light medium, while for the Rev experiment, we grew control cells in heavy, and miRNA cells in light medium.

In solution digestion and isoelectro-focusing of peptide mixtures from the functional experiments (control:miRNA cells)
Equal numbers (12 Â 10 6 ) of heavy and light cells were mixed and lysed in 300 ⎕l RIPA buffer (10 mM Tris-HCl, pH 8.0, 1% Triton, 0.1% SDS, 0.1% Deoxycholate, 140 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche)). Total protein extracts (150 μg) from 1:1 mix of labeled control and miRNA cells were dissolved in denaturation solution (6 M urea, 2 M thiourea in 20 mM ammonium hydrogen carbonate) for 30 min. After complete denaturation, extracts were supplemented with 1 mM DTT and incubated for additional 30 min. Thyols were carboxymethylated with 5 mM IAA for 20 min. Proteins were first digested with 1 μg LysC for 3 h, then diluted 4 Â with 50 mM ammonium bi-carbonate, and digested with 1 μg trypsin overnight at 37°C. The digestion was stopped by acidifying the sample to pH o2 with 100% TFA. We separated peptides according to their isoelectric point (pI) by isoelectrofocusing electrophoresis, using the Agilent 3100 OFFGEL Fractionation Kit (Agilent Technologies) [4], according to the manufacturer's protocol. After separation, peptides mixtures focused at different pH were reconstituted with 1% TFA and desalted and purified on C 18 STAGE tips [5].

In gel digestion of the wt:wt extracts
Protein samples were separated on a 8-12% gradient mini gel (Invitrogen). After Coomassie staining (Colloidal Blue Staining Kit, Invitrogen), each line was cut in fifteen slices and trypsin digested according to a previously described protocol [6]. Briefly, after distaining with 50% acetonitrile (ACN)/25 mM ammonium bicarbonate (NH 4 HCO 3 ) solution, and dehydration by 100% ACN, gel pieces were incubated with 10 mM dithiothreitol in 50 mM NH 4 HCO 3 for 60 min at 56°C for cysteine reduction and then alkylated with 55 mM iodoacetamide in 50 mM NH 4 HCO 3 for 45 min at room temperature, in dark. After several rounds of washings with 50 mM NH 4 HCO 3 and dehydration with 100% ACN, proteins were digested with trypsin overnight, at 37°C. The reaction was stopped by acidification with 2 μl of 50% trifluoroacetic acid (TFA). Peptides were eluted with 30% ACN/3% TFA and 100% ACN. After speed-vacuum centrifugation, peptides were solubilized in 100 μl of 0.1% formic acid (FA), desalted and concentrated using reverse phase C 18 Stage Tips [5]. Peptides were eluted with 80% ACN, lyophilized and re-suspended in 7 μl of 0.1% formic acid for LC-MS/MS analysis.

LC-MS/MS
Peptide mixtures, eluted from C 18 Stage tips, were separated by reversed-phase chromatography on an in-house-made 15 cm column (outer diameter 350 μm, inner diameter 75 μm, 1.9 μm ReproSil, Pur C 18 AQ medium), using a ultra nanoflow high-performance liquid chromatography (HPLC) system (Agilent 1100 Series nano-flow LC system (Agilent Technologies)), directly interfaced to a LTQ-FT Ultra mass spectrometer (Thermo Fisher Scientific). The composition of solvent A was 0.1% FA and 5% ACN in H 2 O and of solvent B was 95% ACN with 0.1% FA. Samples were injected in 0.1% TFA solution at a flow rate of 500 nl/min. The gradient was 140 min, starting from 2% till 60% ACN in 0.5% acetic acid. The spray voltage of a nanoelectrospray ion source (Proxeon) was 1.5-2.0 kV; the temperature was set to 180°C. We used data-dependent mode to automatically switch between MS and MS/MS acquisition. The full scan MS spectra were acquired at a target value of 2,000,000 ions and with a resolution of 100,000 (FWHM) at 400 m/z, while MS/MS spectra were acquired using a target value of 5000 ions and the five most intense ions were isolated for CID-fragmentation.