Computational protocol: Interplay between SIN3A and STAT3 Mediates Chromatin Conformational Changes and GFAP Expression during Cellular Differentiation

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[…] Preparation of Protein Extracts: Cell pellets were first washed three times with PBS and lysed in lysis buffer (0.25 M Tris-HCl at pH 6.8, 1% SDS). Protein concentration was obtained by BCA™ protein assay. Cell lysate was then dried and stored in −30°C.Gel-assisted Digestion : Acrylamide/bisacrylamide (40%, 29∶1), 10% APS, and TEMED were then applied to protein solution to polymerize as a gel directly in the eppendorf. The gel was cut into small pieces and washed several times with TEABC containing 50% ACN. The gel samples were further dehydrated with ACN and the completely dried by vacuum centrifugation. Proteolytic digestion was then performed with trypsin (protein:trypsin = 50∶1, g/g) in 25 mM TEABC with incubation overnight at 37°C and peptides were extracted by 5% FA in 50% ACN, dried in a SpeedVac and stored in −30°C.Immobilized Metal Affinity Chromatography (IMAC) : The IMAC column was first capping one end with a 0.5 µm frit disk enclosed in stainless steel column-end fitting. The Ni-NTA resin was extracted from spin column and packed into a 5-cm microcolumn (500 µm id PEEK column). Automatic purification of phosphopeptides was performed using the IMAC microcolumn connected with autosampler and HP1100 solvent delivery system with a flow rate 13 µl/min. First, the Ni2+ ions were removed with 100 µl EDTA (50 mM) in NaCl (1 M). Then the IMAC column was activated with 100 µl FeCl3 (0.2 M) and equilibrated with loading buffer for 30 min before sample loading. For optimization of the phosphopeptide enrichment, the loading/condition buffer (designated as loading buffer) was 6% (v/v) AA and the pH was adjusted to 3.0 with NaOH (0.1 M at pH 12.8). The peptide samples from trypsin digestion were reconstituted in loading buffer and loaded into the IMAC column that had been equilibrated with the same loading buffer for 20 min. Then the unbound peptides were removed with 100 µl washing solution consisting of 75% (v/v) loading buffer and 25% (v/v) ACN, followed by equilibration with loading buffer for 15 min. Finally, the bound peptides were eluted from the IMAC column with 100 µl NH4H2PO4 (200 mM at pH 4.4). Eluted peptide samples were dried under vacuum and reconstituted in 0.1% (v/v) FA for LC- MS/MS analysis.LC-MS/MS Analysis: Purified phosphopeptide samples were reconstituted in 4 µl buffer A (0.1% FA in H2O) and analyzed by LC-Q-TOF MS (Waters Q-TOFTM Premier from Waters Corp). For LC-MS/MS analysis by Waters Q-TOFTM Premier, samples were injected into a 2 cm×180 µm capillary trap column and separated by 20 cm×75 mm Waters1 ACQUITYTM 1.7 mm BEH C18 column using a nanoACQUITY Ultra Performance LCTM system (Waters Corp). The column was maintained at 35°C and eluted with a linear gradient of 0–80% buffer B (0.1% FA in ACN) for 80, 120, 210 and 270 min. MS was operated in ESI positive V mode with a resolving power of 10,000. NanoLockSpray source was used for accurate mass measurement and the lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a synthetic human [Glu1]-Fibrinopeptide B solution (1 pmol/µl; Sigma) delivered through the NanoLockSpray source. Data acquisition was operated in the data directed analysis (DDA). The method included a full MS scan (m/z 400–1600, 0.6 s) and 3 MS/MS (m/z 100–1990, 1.2 s each scan) sequentially on the most three intense ions present in the full scan mass spectrum.Database Search, Protein Identification and Quantitation: Raw data were processed using Mascot Distiller v (Matrix science). The resulting MS/MS dataset was exported to *mzdata data file format. We performed the peptide identification and assignment of partial post-translational modifications using an in-house version of Mascot v. 2.2 (Matrix science). The datasets were searched against International Protein Index (IPI_human v. 3.29, 68161 sequences) using the following constraints: only tryptic peptides with up to two missed cleavage sites were allowed; 0.3-Da mass tolerances for MS and 0.1-Da mass tolerances for MS/MS fragment ions. Phosphorylation (STY), oxidation (M) was specified as variable modifications. Only unique peptide with scores higher than 25 was confidently assigned. When unique peptides were identified to multiple members of a protein family, proteins having the highest sequence coverage were selected from the Mascot search output result. To evaluate the protein identification false-positive rate, we repeated the searches using identical search parameters and validation criteria against a random database (from the 68161 sequence). Relative quantification of peptides was performed by IDEAL-Q software , . […]

Pipeline specifications

Software tools Mascot Distiller, IDEAL-Q
Databases IPI
Application MS-based untargeted proteomics