Proteomics: Mass spectrometry results from the secretome and cell lysates of iPSC-derived Astrocytes (Progressive Supranuclear Palsy patients and controls)

Description

Mass spectrometry (MS) proteomic protein groups data analysis of the secretome and cell lysates from astrocytes differentiated from iPSC (reprogramed fibroblasts) of Progressive Supranuclear Palsy (PSP) patients (PK01, PK02, PK04, PK08, PK09) and controls (PK03, PK05, PK06, PK12, CQ16, CQ27) in triplicates.

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iPSC- derived astrocytes from PSP patients and controls were cultured (in triplicates) in an astrocyte differentiation medium (AGM, Lonza #3186) for approximately 5 weeks until passage 5. Medium was switched to DMEM/F12 without phenol red (Gibco # 21041-025) without fetal bovine serum (FBS) for 48h. Cells were harvested and pellets were washed 3 times with PBS 1X (pH7.4). Supernatants were collected with PMSF 1 mM and EDTA 1 mM. All samples were frozen at -80°C until the analysis. Proteome sample preparation - Protein digestion and LC-MS/MS analysis In a solution (50 uL) of ammonium bicarbonate 100 mM and urea 8M, we dissolved 10 ug of total proteins of each sample after measurement by BCA. They were reduced with 5 mM dithiothreitol (DTT) for 1h hour at 30 °C, alkylated with 15 mM iodoacetamide for 30 minutes at 30 °C, diluted 10X with 100 mM ammonium bicarbonate, and digested by the addition of two aliquots of trypsin (1:40 and 1:50 w/w, respectively, with an interval of 4 hours between the additions) overnight at 30 °C with constant stirring (400 rpm). Digestion was stopped by the addition of 4% trifluoracetic acid (TFA) and samples were dried. Samples were desalted using the StageTips. Peptides were washed 10 times with 0.1% TFA and subsequently eluted in 50% acetonitrile, and 0.1% TFA. Each sample was injected into an Orbitrap Fusion Lumos mass spectrometer coupled to a Nano EASY-nLC 1200 (Thermo Fisher Scientific). Peptides were injected into a trap column (Thermo Fisher Scientific, nanoViper C18, 3 μm, 75 μm × 2 cm) with 12 µL of solvent A (0.1% formic acid) at 980 bar. After this period, the trapped peptides were eluted onto a C18 column (Thermo Fisher Scientific, nanoViper C18, 2 μm, 75 μm × 15 cm) at a flow rate of 300 nL/min and separated with a gradient of 5-28% acetonitrile with 0.1% formic acid for 80 minutes, followed by 28-40% acetonitrile with formic acid for 10 minutes. Raw files were processed using MaxQuant. The Andromeda algorithm was used for protein identification against the Homo sapiens Uniprot database (downloaded August 2019; 20416 entries). Error mass tolerance for precursors and fragments was set to 4.5 ppm and 0.5 Da, respectively. Cysteine carbamidomethylation was selected as a fixed modification and methionine oxidation and N-terminal acetylation were selected as variable modifications. Trypsin was set as the digestion enzyme, with a maximum of 2 missed cleavages allowed. A maximum false-discovery rate (FDR) of 1% was allowed both for peptides and protein identification. For proteins, it was calculated by using a decoy database created from the reverse ordination of the protein sequences in the Uniprot database. Identification of at least two peptides (unique + razor) was set as a parameter for the identification of a protein. Protein abundances were quantified by the LFQ algorithm. Progressive Supranuclear Palsy patients (PK01, PK02, PK04, PK08, PK09) controls (PK03, PK05, PK06, PK12, CQ16, CQ27)

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